Methods, agents, and compound screening assays for inducing differentiation of undifferentiated mammalian cells into osteoblasts

ABSTRACT

The present invention relates to methods, agents and compound screening assays for inducing differentiation of undifferentiated mammalian cells into osteoblasts. The invention thus provides a method, comprising contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID No: 194-309; and measuring a compound-polypeptide property related to the differentiation of said cells. The invention further relates to a bone formation enhancing pharmaceutical composition, and the use thereof in treating and/or preventing a disease involving a systemic or local decrease in mean bone density in a subject. Furthermore, the invention relates to a method for the in vitro production of bone tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/609,335, filed Oct.30, 2009, which is a divisional of U.S. Ser. No. 11/551,744, filed Oct.23, 2006, issued Nov. 10, 2009 as U.S. Pat. No. 7,615,626, which is acontinuation of PCT/EP2005/051914, filed Apr. 27, 2005, which claimspriority to PCT/EP2004/004522, filed Apr. 27, 2004, both of whichapplications designate the United States, the disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the field of mammalian diseases involving asystemic or local decrease in mean bone density.

BACKGROUND OF THE INVENTION

Bone contains, two distinct cell lineages, i.e. bone-forming cells (e.g.osteoblasts) and bone-resorbing cells (e.g. osteoclasts). Bone is adynamic tissue that is continuously being destroyed (resorbed) andrebuilt, by an intricate interplay between these osteoblasts andosteoclasts. For osteoclasts, a cascade of transcription factors andgrowth factors involved in the progression from progenitor cell tofunctional osteoclast is well established. In contrast, little is knownabout the osteoblast lineage.

Osteoblasts derive from differentiated mesenchymal progenitor cells(MPCs). During the differentiation into osteoblasts bone alkalinephosphatase activity (BAP) becomes upregulated. Bone formation in vivooccurs through two distinct pathways during embryonic development:endochondral or intramembraneous ossification (FIG. 1). As shown in thisfigure, mesenchymal progenitor or stem cells represent the startingpoints for both forms of bone formation. During intramembranousossicification, flat bones such as those of the skull or clavicles, areformed directly from condensations of mesenchymal cells. During theformation of long bones, soon as limb bones, mesenchymal condensationsfirst lead to a cartilage intermediate that is invaded during furtherdevelopment by endothelial cells, osteoclasts and mesenchymal cells thatwill differentiate into osteoblasts and osteocytes (Nakashima and deCrombrugghe, 2003).

A number of diseases are known which are caused by a disturbance of thefine-tuned balance between bone resorption and bone build-up, whichskeletal diseases represent a large number of patients: hypercalcemia ofmalignancy, Paget's disease, inflammatory bone diseases like rheumatoidarthritis and periodontal disease, focal osteogenesis occurring duringskeletal metastases, Crouzen's syndrome, rickets, opsismodysplasia,pycnodysostosis/Toulouse-Lautrec disease, osteogenesis imperfecta, andthe single most important bone disease: osteoporosis.

Currently, osteoporosis affects 1 in 5 women over 50 and 1 in 20 menover 50. For these patients a number of treatments are available, whichmostly tackle the net increase in bone resorption, i.e.:

-   -   hormone replacement therapy (HRT)    -   selective estrogen receptor modulators (SERMs)    -   bisphosphonates    -   calcitonin

While these treatments slow down bone resorption, they do not abolishfracturing because the lost bone is not sufficiently replenished.Fracturing will be stopped when bone formation is sufficientlyincreased. Therefore, there is great interest in identifying osteogenicpathways that lend themselves to therapeutic intervention with boneanabolism as effect. Currently, only one bone anabolic therapy hasreached the osteoporosis market: parathyroid hormone (PTH) 1-34. PTHdisplays bone anabolic effects when administered intermittently. Thetreatment with PTH is, however, very cumbersome because thisbiopharmaceutical needs to be injected daily by the patient. Inaddition, tumor formation has been observed when treating animals athigh doses. Also, it is a very expensive treatment.

Another class of bone anabolics, bone morphogenetic proteins (BMPs),have been approved but only for niche markets, as there aredisadvantages to their use as therapeutic agents to enhance bonehealing. Receptors for the bone morphogenetic proteins have beenidentified in many tissues, and the BMPs themselves are expressed in alarge variety of tissues in specific temporal and spatial patterns. Thissuggests that BMPs may have effects on many tissues other than bone,potentially limiting their usefulness as therapeutic agents whenadministered systemically.

Accordingly, there is a continuing need for novel treatment strategiesand compounds (in particular anabolics) that obviate one or more of thedrawbacks of the currently available treatment strategies.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method for identifyinga compound that induces differentiation of undifferentiated mammaliancells into osteoblasts, comprising contacting a compound with apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID No: 194-309; and measuring a compound-polypeptideproperty related to the differentiation of said cells.

Another aspect of the invention relates to an agent for inducing thedifferentiation of undifferentiated mammalian cells into osteoblasts,selected from the group consisting of an antisense polynucleotide, aribozyme and a small interfering RNA (siRNA), wherein said agentcomprises a nucleic acid sequence complementary to, or engineered from,a naturally occurring polynuoleotide sequence encoding a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID No: 194-309.

A further aspect of the invention relates to a bone formation enhancingpharmaceutical composition comprising a therapeutically effective amountof the agent in admixture with a pharmaceutically acceptable carrier.

Another aspect, of the invention relates to a method for treating and/orpreventing a disease involving a system or local decrease in mean bonedensity an a subject, comprising administering to said subject said boneformation enhancing pharmaceutical composition.

A further a agent of the present invention relates to the use of theabove-described agents in the manufacture of a medicament for thetreatment and/or prevention of a disease involving a systemic or localdecrease in mean bone density.

Another aspect of the invention relates to a method for the in vitroproduction of bone tissue, comprising the steps of contactingundifferentiated mammalian cells with a polynucleotide sequencecomprising a sequence selected from the group consisting of SEQ ID No:1-77 for a time sufficient to differentiate the undifferentiated cellsinto osteoblasts, thereby producing a continuous bone matrix.

DETAILED DESCRIPTION Definitions

The term “carrier” means a non-toxic material used in the formulation ofpharmaceutical compositions to provide a medium, bulk and/or useableform to a pharmaceutical composition. A carrier may comprise one or moreof such materials such as an excipient, stabilizer, or an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude aqueous or solid buffer ingredients including phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone, amino acids such, asglycine, glutamine, asparagine, argintine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counter ions such as sodium; and/or nonionicsurfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

The term “compound” is used herein in the content of a “test compound”or a “drug candidate compound” described in connection with the assaysof the present invention. As such, these compounds comprise organic orinorganic compounds, derived synthetically or form natural sources. Thecompounds include inorganic or organic compounds each aspolynucleotides, lipids or hormone analogs that are characterized byrelatively low molecular weights. Other biopolymeric organic testcompounds include peptides comprising from about 2 to about 40 aminoacids and larger polypeptides comprising from about 40 to about 500amino acids, such as antibodies or antibody conjugates.

The terms: “contact” or “contacting” means bringing at least twomoieties together, whether in an in vitro system or an in vivo system.

The term “condition” or “disease” means the overt presentation ofsymptoms (i.e., illness) or the manifestation of abnormal clinicalindicators (e.g., biochemical indicators). Alternatively, the term“disease” refers to a genetic or environmental risk of or propensity fordeveloping such symptoms or abnormal clinical indicators.

The term “endogenous” shall mean a material that a mammal naturallyproduces. In contrast, the term non-endogenous in this context shallmean that which is not naturally produced by a mammal (for example, andnot limitation, a human) or a virus.

The term “expression” comprises both endogenous expression andoverexpression by transduction.

The term “expressible nucleic acid” means a nucleic acid coding for aproteinaceous molecule, an RNA molecule, or a DNA molecule.

The term “hybridization” means any process by which a strand of nucleicacid binds with a complementary strand through base pairing. The term“hybridization complex” refers to a complex formed between two nucleicacid sequences by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(e.g., C.sub.0t or R.sub.0t analysis) or formed between one nucleic acidsequence present in solution and another nucleic acid, sequenceimmobilized on a solid support, (e.g., paper, membranes, filters, chips,pins or glass slides, or any other appropriate substrate to which cellsor their nucleic acids have been fixed). The term “stringent conditions”refers to conditions that permit hybridization between polynucleotidesand the claimed polynucleotides. Stringent conditions can be defined bysalt concentration, the concentration of organic solvent, e.g.,formamide, temperature, and other conditions well known is the art. Inparticular, reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature canincrease stringency.

The term “inhibit” or “inhibiting”, in relationship to the term“response” means that a response is decreased or prevented in thepresence of a compound, as opposed to in the absence of the compound.

The term “pharmaceutically acceptable prodrugs” as used herein means theprodrugs of the compounds useful in the present invention, which are,within line scope of sound medical judgment, suitable for use to contactwith the tissues of patients with undue toxicity, irritation, allergicresponse commensurate with a reasonable benefit/risk ratio, andeffective for their intended use of the compounds of the invention. Theterm “prodrug” means a compound that is transformed in vivo to yield aneffective compound useful in the present invention or a pharmaceuticallyacceptable salt, hydrate or solvate thereof. The transformation mayoccur by various mechanisms, such as through hydrolysis in blood. Thecompounds bearing metabolically cleavable groups have the advantage thatthey may exhibit improved bioavailability as a result of enhancedsolubility and/or rate of absorption conferred open the parent compoundby virtue of the presence of the metabolically cleavable group, thus,such compounds act as pro-drugs. A thorough discussion is provided inDesign of Prodrugs, H. Bundgaard, ed., Elsevier (1985); Methods inEnzymology; K. Widder et al, Ed., Academic Press, 42, 309-396 (1985); ATextbook of Drug Design and Development, Krogsgaard-Larsen and H.Bandaged, ed., Chapter 5; “Design and Applications of Prodrugs” 113-191(1991); Advanced Drug Delivery Reviews, H. Bundgard, 8, 1-38, (1992); J.Pharm. Sci., 77, 285 (1988); Chem. Pharm. Bull., N. Nakeya et al, 32,692 (1984); Pro-drugs as Novel Delivery Systems, T. Higuchi and V.Stella, 14 A.C.S. Symposium Series, and Bioreversible Carriers In DrugDesign, E. B. Roche, ed., American Pharmaceutical Association andPergamon Press, 1987, which are incorporated herein by reference. Anexample of the prodrugs in an ester prodrug. “Ester prodrug” means acompound that is convertible in vivo by metabolic means (e.g., byhydrolysis) to an inhibitor compound according to the present invention.For example an ester prodrug of a compound containing a carboxy groupmay be convertible by hydrolysis in vivo to the corresponding carboxygroup.

The term “pharmaceutically acceptable salts” refers to the non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of compounds useful in thepresent invention.

The term “polynucleotide” means a polynucleic acid, in single or doublestranded form, and in the sense or antisense orientation, complementarypolynucleic acids that hybridize to a particular polynucleic acid understringent conditions, and polynucleotides that are homologous in atleast, about 60 percent of its base pairs, and more preferably 70percent of its base pairs are in common, most preferably 90 percent, andin a special embodiment 100 percent of its base pairs. Thepolynucleotides include polyribonucleic acids, polydeoxyribonucleicacids, and synthetic analogues thereof. The polynucleotides aredescribed by sequences that vary it length, that range from about 10 toabout 5000 bases, preferably about 100 to about 4000 bases, morepreferably about 250 to about 2500 bases. A preferred polynucleotideembodiment comprises from about 10 to about 30 bases in length. Aspecial embodiment of polynucleotide is the polyribonucleotide of fromabout 10 to about 22 nucleotides, more commonly described as smallinterfering RNAs (siRNAs). Another special embodiment are nucleic acidswith modified backbones such as peptide nucleic acid (PNA),polysiloxane, and 2′-O-(2-methoxy)ethylphosphorothioate, or includingnon-naturally occurring nucleic acrid residues, or one or more nucleicacid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,amino-, propyl-, chloro-, and methanocarbanucleosides, or a reportermolecule to facilitate its detection.

The term “polypeptide” relates to proteins, proteinaceous molecules,fractions of proteins peptides and oligopeptides.

The term “solvate” means a physical association of a compound useful inthis invention with one or more solvent molecules. This physicalassociation includes hydrogen bonding. In certain instances the solvatewill be capable of isolation, for example when one or more solventmolecules are incorporated in the crystal lattice of the crystallinesolid. “Solvate” encompasses both solution-phase and isolable solvates.Representative solvates include hydrates, ethanolates and methanolates.

The term “subject” includes humans and other mammals.

The term “effective amount” or “therapeutically effective amount” meansthat amount of compound, or agent that will elicit the biological ormedical response of a subject that is being sought, by a medical doctoror other clinician. In particular, with regard to treating abone-related disorder, the term “effective amount” is intended to meanthat amount of a compound or agent that will elicit the biological ormedical response of a subject that is being sought by a medical doctoror other clinician, in particular, with regard to treating a conditioninvolving a systemic of local decrease in mean bone density, the term“effective amount” is intended to mean that effective amount of ancompound or agent that will bring about a biologically meaningfulincrease in mean bone density.

The term “treating” means an intervention performed with the intentionof preventing the development or altering the pathology of, and therebyalleviating a disorder, disease or condition, including sue or moresymptoms of such disorder or condition. Accordingly, “treating” refersto both therapeutic treatment and prophylactic or preventative measures.Those in need of treating include those already with the disorder aswell as those in which that disorder is to be prevented. The relatedterm “treatment,” as used herein, refers to the act of treating adisorder, symptom, disease or condition, as the term “treating” isdefined above.

“Undifferentiated mammalian cells” are pluropotent cells which are in anearly stage of specialization, i.e. cells which do not yet have theirfinal function and can be induced to form almost any given cell type. Inparticular, these are cells which have not yet differentiated to thespecific bone cells osteoblasts or osteoclasts. Such pluropotent cellsare especially blood cells and cells present in bone marrow, as well ascells derived from adipose tissue. In addition, cells which can bedifferentiated into mesenchymal precursor cells are contemplated in thepresent invention, such as, for example, totipotent stem cells such asembryonic stem cells.

One aspect of the present invention is related to a method foridentifying a compound that induces differentiation of undifferentiatedmammalian cells into osteoblasts, comprising contacting a compound witha polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID No: 194-309; and measuring a compound-polypeptideproperty related to the differentiation of said cells. The“compound-polypeptide property” is a measureable phenomenon chosen bythe person of ordinary skill in the art. The measurable property maye.g. be the binding affinity for a peptide domain of the polypeptide orthe level of any one of a number of biochemical marker levels ofosteoblast differentiation. Osteoblast differentiation can e.g. bemeasured by measuring the level of enzymes that are induced during thedifferentiation process, such as alkaline phosphatase, type-1 collagen,osteocalcin and osteopontin. The alkaline phosphatase activity can foemeasured by adding methylumbelliferyl heptaphosphate (MUP) solution(Sigma) to the cells. The fluorescence generated upon cleavage of theMUP substrate by the AP activity is measured on a fluorescence platereader (Fluostar, BMG).

In a preferred embodiment of the invention, tint polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ ID No:199, 230, 237, 262 and 281 (Table 2B).

Depending on the choice of the skilled artisan, the present assay methodmay be designed to function as a aeries of measurements, each of whichto designed to determine whether the drug candidate compound is indeedacting on the polypeptide to thereby induce the differentiation ofundifferentiated cells into osteoblasts. For example, an assay designedto determine the binding affinity of a compound to the polypeptide, orfragment thereof, may be necessary, but not sufficient, to ascertainwhether the test compound would be useful for increasing mean bonedensity when administered to a subject. Nonetheless, such bindinginformation would be useful in identifying a set of test compounds foruse in an assay that would measure a different property, further downthe biochemical pathway, such as bone mineralization, assayed bymeasuring the amount of deposited calcium. Such second assay may bedesigned to confirm that the test compound, having binding affinity forthe polypeptide, actually induces the differentiation ofundifferentiated cells into osteoblasts. Suitable controls should alwaysbe in place to insure against false positive readings.

The order of taking these measurements is not believed to be critical tothe practice of the present invention, which may be practiced in anyorder. For example, one may first perform a screening assay of a set ofcompounds for which no information is known respecting the compounds'binding affinity for the polypeptide. Alternatively, one may screen aset of compounds identified as having binding affinity for a polypeptidedomain, or a class of compounds identified as being an inhibitor of thepolypeptide. However, for the present assay to be meaningful to theultimate use of the drug candidate compounds, a measurement of bonealkaline phosphatase levels or bone mineralization is necessary.Validation studies including controls, and measurements of bindingaffinity to the polypeptides of the invention are nonetheless useful inidentifying a compound useful in any therapeutic or diagnosticapplication.

The present assay method may be practiced, in a cell-free system invitro, using one or more of the polypeptides, or fragments thereof. Thebinding affinity of the compound with the polypeptide can be measured bymethods known in the art, such as using surface plasmon resonancebiosensors (Biacore), by saturation binding analysis with a labeledcompound (e.g. Scatchard and Lindmo analysis), by differential UVspectrophotometer, fluorescence polarization assay, Fluorometric ImagingPlate Reader (FLIPRO) system, Fluorescence resonance energy transfer,and Bioluminescence resonance energy transfer. The blinding affinity ofcompounds can also be expressed in dissociation constant (Kd) or as IC50or EC50. The IC50 represents the concentration of a compound that isrequired for 50% inhibition of binding of another ligand to thepolypeptide. The EC50 represents the concentration required forobtaining 50% of the maximum affect in any assay that measures receptorfunction. The dissociation constant, Kd is a measure of how well aligand binds to the polypeptide, it is equivalent to the ligandconcentration required to saturate exactly half of the binding-sites onthe polypeptide. Compounds with a high affinity binding have low Kd,IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate tolow affinity binding relates to a high Kd, IC50 and EC50 values, i.e. inthe micromolar range.

The present assay method may also be practiced in a cellular assay. Ahost cell expressing the polypeptide can be a cell with endogenousexpression or a cell over-expressing the polypeptide by transduction.When the endogenous expression of the polypeptide is not sufficient todetermine a baseline that can easily be measured, one may use using hostcells that over-express the polypeptide. In such cellular assay, thebiological activity of the polypeptide may be measured by following theproduction of bone alkaline phosphatase (BAP) or bone mineralization.

The present invention further relates to a method for identifying acompound that induces differentiation of undifferentiated mammaliancells into osteoblasts, comprising:

-   -   (a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        1984-309;    -   (b) determining the binding affinity of the compound to the        polypeptide;    -   (c) contacting a population of mammalian ceils expressing said        polypeptide with the compound that exhibits a binding affinity        of at least 10 micromolar; and    -   (d) identifying the compound that induces the differentiation of        said undifferentiated cells.

For high-throughput purposes, libraries of compounds may be used such asantibody fragment libraries, peptide phage display libraries, peptidelibraries (e.g. LOPAP™, Sigma Aldrich), lipid libraries (BioMol),synthetic compound libraries (e.g. LOPAC™, Sigma Aldrich) or naturalcompound libraries (Specs, TimTec).

Preferred drug candidate compounds are low molecular weight compounds.Low molecular weight compounds, i.e. with a molecular weight ob 500Dalton or less, are likely to have good absorption and permeation inbiological systems and are consequently more likely to be successfuldrug candidates than compounds with a molecular weight above 500 Dalton.Peptides comprise another preferred class of drug candidate compounds.Peptides may be excellent drug candidates and there are multipleexamples of commercially valuable peptides such as fertility hormonesand platelet aggregation inhibitors. Natural compounds are anotherpreferred class of drug candidate compound. Such compounds are found inand extracted from natural sources, and which may thereafter besynthesized. The lipids are another preferred class of drug candidatecompound.

Another preferred class of drug candidate compounds is an antibody. Thepresent invention also provides antibodies directed against theextracellular domains of the polypeptides of the invention. Theseantibodies should specifically bind to one or more of the extra-cellulardomains of the polypeptides, or as described further below, engineeredto be endogenously produced to bind to an intra-cellular polypeptidedomain. These antibodies may be monoclonal antibodies or polyclonalantibodies. The present invention includes chimeric, single chain, andhumanized antibodies, as well as FAb fragments and the products of a FAbexpression library, and Fv fragments and the products of an Fvexpression library.

In certain embodiments, polyclonal antibodies may be used in thepractice of the invention. The skilled artisan knows methods ofpreparing polyclonal antibodies. Polyclonal antibodies can be raised ina mammal, for example, by one or more injections of an immunizing agentand, if desired, an adjuvant. Typically, the immunizing agent and/oradjuvant will be injected in the mammal by multiple subcutaneous orintraperitoneal injections. Antibodies may also be generated against theintact protein or polypeptide, or against a fragment such as itsextracellular domain peptides, derivatives including conjugates, orother epitope of the protein or polypeptide, such as the polypeptideembedded in a cellular membrane, or a library of antibody variableregions, such as a phage display library.

It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants that may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). One skilled in the art withoutundue experimentation may select the immunization protocol.

In some embodiments, the antibodies may be monoclonal antibodies.Monoclonal antibodies may be prepared using methods known in the art.The monoclonal antibodies of the present invention may be “humanised” toprevent the host from mounting an immune response to the antibodies. A“humanized antibody” is one in which the complementarity determiningregions (CDRs) and/or other portions of the light and/or heavy variabledomain framework are derived from a non-human immunoglobulin, but theremaining portions of the molecule are derived from one or more humanimmunoglobulins. Humanized antibodies also include antibodiescharacterized by a humanized heavy chain associated with a donor oracceptor unmodified light chain or a chimeric light chain, or viceversa. The humanization of antibodies may be accomplished by methodsknown in the art (see, e.g. Mark and Padlan, (1994) “Chapter 4.Humanization of Monoclonal Antibodies”, The Handbook, of ExperimentalPharmacology Vol. 113, Springer-Verlag, New York), Transgenic animalsmay be used to express humanised antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter,(1991) J. Mol. Biol. 227:381-8; Marks et al. (1991). J. Mol. Biol.222:581-97), The techniques of Cole, et. al, and Boerner, et al. arealso available for the preparation of human monoclonal antibodies (Cole,et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R, Liss, p.77; Boerner, et al (1991). J. Immunol., 147(1):86-95).

Techniques known in the art for the production of single chainantibodies can be adapted to produce single chain antibodies to thepolypeptides and proteins of that present invention. The antibodies maybe monovalent antibodies. Methods for preparing monovalent antibodiesare well known in the art. For example, one method involves recombinantexpression of immunoglobulin light chain and modified heavy chain. Theheavy chain is truncated generally at any point in that Fc region so asto prevent heavy chain cross-linking. Alternatively; the relevantcysteine residues are substituted with another amino acid residue or aredetected so as to prevent cross-linking.

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens and preferably for a cell-surface protein or receptor orreceptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, (1983) Nature 305:537-9). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. Affinitychromatography steps usually accomplish the purification of the correctmolecule. Similar procedures are disclosed in Trauneeker, et al. (1991)EMBO J. 10:3655-9.

According to another preferred embodiment, that assay method comprisesusing a drug candidate compound identified as having a binding affinityfor a polypeptide comprising an amino acid sequence selected from thegroup consisting of 194-309.

The invention further relates to an agent for inducing thedifferentiation of undifferentiated a mammalian cells into osteoblasts,selected from the group consisting of an antisense polynucleotide, aribozyme, and a small interfering RNA (siRNA), wherein said agentcomprises a nucleic acid sequence complementary to, or engineered from,a naturally-occurring polynucleotide sequence encoding a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO; 194309. In a preferred embodiment, the agent is selected fromthe group consisting of an antisense polynucleotide, a ribozyme, and asmall interfering RNA (siRNA), wherein said agent comprises a nucleicacid sequence complementary to, or engineered from, anaturally-occurring polynucleotide sequence encoding a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 199, 230, 237, 262 and 181.

One embodiment of the agent is a nucleic acid that is antisense to anucleic acid comprising SEQ ID NO: 83-193. Preferably, the agent is anucleic acid that is antisense to a nucleic acid comprising SEQ IDNo:83, 114, 121, 146, and 165. For example, an antisense nucleic acid(e.g. DNA) may be introduced into cells in vitro, or administered to asubject in vivo, as gene therapy to inhibit cellular expression ofnucleic acids comprising SEQ ID NO: 78-193, preferably comprising SEQ IDNO 199, 230, 237, 262 and 281. Antisense oligonucleotides preferablycomprise a sequence containing from about 17 to about 100 nucleotidesand more preferably the antisense oligonucleotides comprise from about18 to about 30 nucleotides. Antisense nucleic acids may be prepared fromabout 10 to about 30 contiguous nucleotides selected from the sequencesof SEQ ID NO: 78-193, expressed in the opposite orientation.

The antisense nucleic acids are preferably oligonucleotides and mayconsist entirely of deoxyribo-nucleotides, modifieddeoxyribonucleotides, or some combination of both. The antisense nucleicacids can be synthetic oligonucleotides. The oligonucleotides may bechemically modified, if desired, to improve stability and/orselectivity. Since oligonucleotides are susceptible to degradation byintracellular nucleases, the modifications can include, for example, theuse of a sulfur group to replace the free oxygen of the phosphodiesterbond. This modification is called a phosphorothioate linkage.Phosphorothioate antisense oligonucleotides are water soluble,polyanionic, and resistant to endogenous nucleases. In addition, when aphosphorothioate antisense oligonucleotide hybridizes to its targetsite, the RNA-DNA duplex activates the endogenous enzyme ribonuclease(RNase) H, which cleaves the mRNA component of the hybrid molecule.

In addition, antisense oligonucleotides with phosphoramidite andpolyamide (peptide) linkages can be synthesized. These molecules shouldbe very resistant to nuclease degradation. Furthermore, chemical groupscan be added to the 2′ carbon of the sugar moiety and the 5 carbon (C-5)of pyrimidines to enhance stability and facilitate the binding of theantisense oligonucleotide to its target site. Modifications may include2′-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxyphosphorothioates, modified bases, as well as other modifications knownto those of skill in the art.

Another type of expression-inhibitory agent that reduces the levels ofthe polypeptides of the invention is ribozymes. Ribozymes are catalyticRNA molecules (RNA enzymes) that have separate catalytic and substratebinding domains. The substrate binding sequence combines by nucleotidecomplementarity and, possibly, non-hydrogen bond interactions with itstarget sequence. The catalytic portion cleaves the target RNA at aspecific site. The substrate domain of a ribozyme can be engineered todirect it to a specified mRNA sequence. The ribozyme recognizes and thenbinds a target mRNA through complementary base pairing. Once it is boundto the correct target site, the ribozyme acts enzymatically to cut thetarget mRNA. Cleavage of the mRNA by a ribozyme destroys its ability todirect synthesis of the corresponding polypeptide. Once the ribozyme hascleaved its target sequence, it is released and can repeatedly bind andcleave at other mRNAs.

Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitisdelta virus, group I intron or RNaseP RNA (in association with an RNAguide sequence) motif or Neurospora VS RNA motif. Ribosymes possessing ahammerhead or hairpin structure are readily prepared since thesecatalytic RNA molecules can be expressed within ceils from eukaryoticpromoters (Chen, et al. (1992) Nucleic Acids Re. 20:4581-9). A ribozymeof the present invention can be expressed in eukaryotic cells from theappropriate DNA vector. If desired, the activity of the ribozyme may beaugmented by its release from the primary transcript by a secondribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21:3249-55).

Ribozymes may be chemically synthesized by combining anoligodeoxyribonucleotide with a ribozyme catalytic domain (20nucleotides) flanked by sequences that hybridize to the target mRNAafter transcription. The oligodeoxyribonucleotide is amplified by usingthe substrate binding sequences as primers. The amplification produce iscloned into a eukaryotic expression vector.

Ribozymes are expressed from transcription units inserted into DNA, RNA,or viral vectors. Transcription of the ribozyme sequences a drivers froma promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase II(pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters will be expressed at high levels in all cells; thelevels of a given pol II promoter in a given cell type will depend onnearby gene regulatory sequences. Prokaryotic RNA polymerase promotersare also used, providing that the prokaryotic RNA polymerase enzyme isexpressed in the appropriate cells (Gao and: Huang, (1993) Nucleic AcidsRes. 21:2867-72). It has been demonstrated that, ribozymes expressedfrom these promoters can function in mammalian cells (Kashani-Sabet, etal. (1992) Antisense Res. Dev. 2:3-15).

A particularly preferred inhibitory agent is a small interfering RNA(siRNA). SiRNAs mediate the post-transcriptional process of genesilencing by double stranded RNA (dsRNA) that is homologous in sequenceto the silences RNA. SiRNA according to the present invention comprisesa sense strand of 17-25 nucleotides complementary or homologous to acontiguous 17-25 nucleotide sequence selected from the group ofsequences described in SEQ ID NO: 78-193, preferably from the group ofsequences described in SEQ ID No: 83, 114, 121, 146 and 165 and anantisense strand of 17-23 nucleotides complementary to the sense,strand. The most preferred siRNA comprises sense and antisense strandsthat are 100 percent complementary to each other and the targetpolynucleotide sequence. Preferably the siRNA further comprises a loopregion linking the sense and the antisense strand.

A self-complementing single stranded siRNA molecule polynucleotideaccording to the present invention comprises a sense portion and anantisense portion connected by a loop region linker. Preferably, theloop region sequence is 4-30 nucleotides long, more preferably 5-15nucleotides long and most preferably 8 nucleotides long. In a mostpreferred embodiment the linker sequence is GTTTGCTATAAC as identifiedby SEQ ID No. 310. Self-complementary single stranded siRNAs formhairpin loops and are more stable than ordinary dsRNA. In addition, theyare more easily produced from vectors.

Analogous to antisense RNA, the siRNA can be modified to confirmresistance to nucleolytic degradation, or to enhance activity, or toenhance cellular distribution, or to enhance cellular uptake, soonmodifications may consist of modified internucleoside linkages, modifiednucleic acid bases, modified sugars and/or chemical linkage the SiRNA toone or more moieties or conjugates. The nucleotide sequences areselected according so siRNA designing rules that gave an improvedreduction of the target sequences compared to nucleotide sequences thatdo not comply with these siRNA designing roles (For a discussion ofthese rules and examples of the preparation of siRNA, WO2004094636,published Nov. 4, 2004, and UA20030198627, are hereby incorporated byreference.

The present invention also relates to compositions, and methods usingsaid compositions, comprising a DNA expression vector capable ofexpressing a polynucleotide capable of inducing differentiation ofundifferentiated cells into osteoblasts and described hereinabove as anexpression inhibition agent.

The polynucleotide expressing the expression-inhibiting agent ispreferably included within a vector. The polynucleic acid is operablylinked to signals enabling expression of the nucleic acid sequence andis introduced into a cell utilizing, preferably, recombinant vectorconstructs, which will express the antisense nucleic acid once thevector is introduced into the cell. A variety of viral-based systems areavailable, including adenoviral, retroviral, adeno-associated viral,lentiviral, herpes simplex viral or a sendaviral vector systems, and allmay be used to introduce and express polynucleotide sequence for theexpression-inhibiting agents in target cells.

Preferably, the viral vectors used in that methods of the presentinvention are replication defective. Such replication defective vectorswill usually pact at least one region that is necessary for onereplication of the virus in the infected, cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution, partial deletion or additionsof one or more bases to an essential (for replication) region. Such,techniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Preferably, the replication defective virus retainsthe sequences of its genome, which are necessary for encapsidating, theviral particles.

In a preferred embodiment, the viral element is derived from anadenovirus. Preferably, the vehicle includes an adenoviral vectorpackaged into an adenoviral capsid, or a functional part, derivative,and/or analogue thereof. Adenovirus biology is also comparatively wellknown on the molecular level. Many tools for adenoviral vectors havebeen and continue to be developed, thus making an adenoviral capsid apreferred vehicle for incorporating in a library of the invention. Anadenovirus is capable of infecting a wide variety of cells. However,different adenoviral serotypes have different preferences for cells. Tocombine and widen the target cell population that an adenoviral capsidor the invention can enter in a preferred embodiment, the vehicleincludes adenoviral fiber proteins from at least two adenoviruses.

In a preferred embodiment, the nucleic acid derived from an adenovirusincludes the nucleic acid encoding an adenoviral late protein or afunctional part, derivative, and/or analogue thereof. An adenoviral lateprotein, for instance an adenoviral fiber protein, may be favorably usedto target the vehicle to a certain cell or to induce enhanced deliveryof the vehicle to the cell. Preferably, the nucleic acid derived from anadenovirus encodes for essentially all adenoviral late proteins,enabling the formation of entire adenoviral capsids or functional parts,analogues, and/or derivatives thereof. Preferably, the nucleic acidderived from an adenovirus includes the nucleic acid encoding adenovirusE2A or a functional part, derivative, and/or analogue thereof.Preferably, the nucleic acid derived from an adenovirus includes thenucleic acid encoding at least one E4-region protein or a functionalpart, derivative, and/or analogue thereof, which facilitates, at leastin part, replication of an adenoviral derived nucleic acid in a cell.The adenoviral vectors used in the examples of this application areexemplary of the vectors useful in the present method of treatmentinvention.

Certain embodiments of the present invention use retroviral vectorsystems. Retroviruses are integrating viruses that infect dividingcells, and their construction is known in the art. Retroviral vectorscan be constructed from different types of retrovirus, such as, MoMuLV(“murine Moloney leukemia virus” MSV (“murine Moloney sarcoma virus”),HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Roussarcoma virus”) and Friend virus. Lentiviral vector systems may also beused in the practice of the present invention. Retroviral systems andherpes virus system may be preferred, vehicles for transfection ofneuronal cells.

In other embodiments of the present invention, adeno-associatedassociated viruses (“AAV”) are utilised. The AAV viruses are DNA virusesof relatively small size that integrate, in a stable and site-specificmanner, into the genome of the reflected cells. They are able to infecta wide spectrum of cells without inducing any effects on cellulargrowth, morphology or differentiation, and they do not appear to beinvolved in human pathologies.

In the vector construction, the polynucleotide agents of the presentinvention may be linked to one or more regulatory regions. Selection ofthe appropriate regulatory region or regions is a routine matter, withinthe level of ordinary skill in the art. Regulatory regions includepromoters, and may include enhancers, suppressors, etc.

Promoters that may be used in the expression vectors of the presentinvention include both constitutive promoters and regulated (inducible)promoters. The promoters may be prokaryotic or eukaryotic depending onthe host. Among the prokaryotic (including bacteriophage) promotersuseful for practice of this invention are lac, lacZ, T3, T7, lambdaP.sub.r, P.sub.1, and trp promoters. Among the eukaryotic (includingviral) promoters useful for practice of this invention are ubiquitouspromoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filamentpromoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic genepromoters (e.g. MDR, type, CFTR, factor VIII), tissue-specific promoters(e.g. actin promoter in smooth muscle cells, or Fit and Flk promotersactive in endothelial cells), including animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals; elastase I gene control region which is active inpanereatic acinar cells (Swift, et al. (1984) Cell 38:639-46; Ornitz, etal. (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,(1987) Hepatology 7:425-515); insulin gene control region which isactive in pancreatic beta cells (Hanahan, (1985) Nature 315:115-22),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mousemammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95),albumin gene control region which is active in liver (Pinkert, et al.(1985) Genes and Devel. 1:268-76), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf, et al. (1985) Mol. Cell. Biol.,5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1-antitrypsingene control region which is active in the liver (Kelsey, et al. (1987)Genes and Devel., 1: 161-71), beta-globin gene control region which isactive in myeloid ceils (Mogram, et al. (1985) Nature 315:338-40;Kollias, et al. (1986) Cell 46:89-94), myelin basic protein gene controlregion which is active in oligodendrocyte cells in the brain (Readhead,et al. (1987) Cell 48:703-12), myosin light chain-2 gene control regionwhich is active in skeletal muscle (Sani, (1985) Nature 314.283-6), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason, et. al. (1986) Science 234:1372-8).

Other promoters which may used in the practice of the invention includepromoters which are preferentially activated in dividing cells,promoters which respond to a stimulus (e.g. steroid hormone receptor,retinoic acid receptor), tetracycline-regulated transcriptionalmodulators, cytomegalovirus immediate-early, retroviral LTR,metallothionein, SV-40 Ela, and MLP promoters.

Additional vector systems include the non-viral systems that facilitateintroduction of polynucleotide agents into a patient. For example, a DNAvector encoding a desired sequence can be introduced in vivo bylipofection. Synthetic cationic lipids designed to limit thedifficulties encountered with liposome-mediated transfection can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner, et. al. (1987) Proc. Natl. Acad Sci. USA 84:7413-7);see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:802731; Ulmer,et al. (1993) Science 259:1745-8). The use of cationic lipids maypromote encapsulation of negatively charged nucleci acids, and alsopromote fusion with negatively charged cell membranes. Felgner andRingold, (1989) Nature 337:387-8). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO 95/18863 and WO 96/17823, and inU.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenousgenes into the specific organs in vivo has certain practical advantagesand directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, forexample, pancreas, liver, kidney, and the brain. Lipids may bechemically coupled to other molecules for the purpose of targeting.Targeted peptides, e.g., hormones or neurotransmiters, and proteins forexample, antibodies, or non-peptide molecules could be coupled toliposomes chemically. Other molecules are also useful for facilitatingtransfection of a nucleic acid in vivo, for example, a cationicoligopeptide (e.g., International Patent Publication WO 95/21931),peptides derived from DNA binding proteins (e.g., International PatentPublication WO 96/25508), or a cationic polymer (e.g., InternationalPatent Publication WO 95/21931).

It is also possible to introduce a DNA vector in vivo as a naked DNAplasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). NakedDNA vectors for therapeutic purposes can be introduced into the desiredhost cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem.267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al.Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30).Receptor-mediated DNA delivery approaches can also be used (Curiel, etal. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem.262:4429-32).

The invention further provides is a bone formation enhancingpharmaceutical composition comprising a therapeutically effective amountof an agent as described hereinabove, in admixture with apharmaceutically acceptable carrier. Another preferred embodiment ispharmaceutical composition for the treatment or prevention of acondition involving a systemic or local decrease in mean bone density,or a susceptibility to the condition, comprising an effective boneformation enhancing amount of antagonists or inverse agonists of thepolypeptides of the invention and/or pharmaceutically acceptable salts,hydrates, solvates, or prodrugs thereof in admixture with apharmaceutically acceptable carrier.

The pharmaceutical composition may be composition, that may be solid,liquid, gel, or other form, in which the compound, polynucleotide,vector, and antibody of the invention is maintained in an active form,e.g., in a form able to effect a biological activity. For example, acompound of the invention would have inverse agonist or antagonistactivity on the polypeptide; a nucleic acid would be able to replicate,translate a message, or hybridize to a complementary mRNA of thepolypeptide; a vector would be able to transfect a target cell andexpression the antisense, antibody, ribozyme or siRNA as describedhereinabove; an antibody would bind a polypeptide domain.

Such compositions can be formulated for administration by topical, oral,parenteral, intranasal, subcutaneous, and intraocular, routes.Parenteral administration is meant to include intravenous injection,intramuscular injection, intraarterial injection or infusion techniques.The composition may be administratered parenterally indosage unitformulations containing standard, well-known non-toxic physiologicallyacceptable carriers, adjuvants and vehicles as desired.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, foringestion by the patient. Pharmaceutical compositions for oral use canbe prepared by combining active compounds with solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulsoe, such as methyl cellulose, hydorxypropylmethyl-cellulose, orsodium carboxymethyl-cellulose; gums including arabic and tragacanth;and proteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate. Dragee cores may be used in conjunction with suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification or to characterizethe quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Preferred sterile injectable preparations can be a solution orsuspension in a non-toxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, buffered,saline, isotonic saline (e.g., monosodium or disodium phosphate, sodium,potassium; calcium or magnesium chloride, or mixtures of such salts).Ringer's solution, dextrose, water, sterile water, glycerol, ethyanol,and combinations thereof 1,3-butanediol and sterile fixed oils areconveniently employed as solvents or suspending media, Any bland fixedoil can be employed, including synthetic mono- or di-glycerides. Fattyacids such as oleic acid also find use in the preparation ofinjectables.

The composition medium can also be a hydrogel, which is prepared fromany biocompatible or non-cytotoxic homo- or hetero-polymer, such as ahychrophilic polyaorylic acid polymer that can act as a drug absorbingsponge. Certain of them, such as, in particular, shore obtained fromethylene and/or propylene oxide are commercially available, A hydrogelcan be deposited directly onto the surface of the tissue to be treated,for sample during surgical intervention.

Embodiments of pharmaceutical compositions of the present inventioncomprise a replication defective recombinant viral vector encoding thepolynucleotide inhibitory agent of the present invention and atransfaction enhancer, such as poloxamer. An example of a poloxamer isPoloxamer 407, which is commercially available (BASF, Parsippany, N.J.)and is a non-toxic, biocompatible polyol. A polyoxamer impregnated withrecombinant viruses may be deposited directly on the surface of thetissue to be treated, for example during a surgical intervention.Poloxamer possesses essentially the same advantages as hydrogel whilehaving a lower viscosity.

The active expression-inhibiting agent may also be entrapped inmicrocapsules prepared, for example, by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methlymethacylate) mocrocapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-articles and nanocapsules) or in macroemulsion.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980) 16th edition, Osol, A. Ed.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples oilsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™. (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-Hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

As defined above, therapeutically effective dose means that amount ofprotein, polynucleotide, peptide, or its antibodies, agonists orantagonists, which ameliorate the symptoms or condition. Therapeuticefficacy and toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED50 (the dose therapeutically effective in 50% of the population)and LD50 (the dose lethal to 50% of the population). The dose ration oftoxic to therapeutic effects is the therapeutic index, and it can beexpressed as the ratio, LD50/ED50. Pharmaceutical compositions thatexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies is used in formulating a range ofdosage for human use. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, dragcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The pharmaceutical compositions according to this invention may beadministered to a subject by a variety of methods. They may be addeddirectly to target tissues, complexed with cationic lipids, packagedwithin liposomes, or delivered to target cells by other methods known inthe art. Localised administration to the desired tissues may be done bycatheter, infusion pump or stent. The DNA, DNA/vehicle complexes, or therecombinant virus particles are locally administered to the site oftreatment. Alternative routes of delivery include, but are not limitedto, intravenous injection, intramuscular injection, subcutaneousinjection, aerosol inhalation, oral (tablet or pill form), topical,systemic, ocular, intraperitoneal and/or intrathecal delivery. Examplesof ribozyme delivery and administration are provided in Sullivan et al.WO 94/02595.

Antibodies according to the invention may be delivered as a bolus only,infused over time or both administered as a bolus and infused over time.Those skilled in the art may employ different formulations forpolynucleotides than for proteins. Similarly, delivery ofpolynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

As discussed hereinabove, recombinant viruses may be used to introduceDNA encoding polynucleotide agents useful in the present invention.Recombinant viruses according to the invention are generally formulatedand administered in the form of doses of between about 10.sup.4 andabout 10.sup.14 pfu. In the case of AAVs and adenoviruses, doses of fromabout 10.sup.6 to about 10.sup.11 pfu are preferably sued. The term pfu(“plaque-forming unit”) corresponds to the infective power of asuspension of virions and is determined by infecting an appropriate cellculture and measuring the number of plaques formed. The techniques fordetermining the pfu titre of a viral solution are well documented in theprior art.

A further aspect of the invention relates to a method of treating orpreventing a disease involving a systemic or local descrease in meanbone density, comprising administering to said subject a bone formationenhancing pharmaceutical composition as described herein.

The invention also relates to the use of an agent as described above forthe preparation of a medicament for treating or preventing a diseaseinvolving a systemic or local descrease in mean bone density.

In a preferred embodiment of the present invention the disease isselected from the group consisting of osteoporosis, hypocalcaemia ofmalignancy, multiple myelomatosis, hyperparathyroidism, andhyperthyroidism. A special embodiment of this invention is a methodwherein the disease is osteoporosis.

Still another aspect or the invention relates to a method for diagnosinga pathological condition involving a systemic or local decrease in meanbone density or a susceptibility to the condition in a subject,comprising determining the amount of polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 194-309in a biological sample, and comparing the amount with the amount of thepolypeptide in a healthy subject, wherein an increase of the amount ofpolypeptide compared to the healthy subject is indicative of thepresence of the pathological condition.

Preferably the pathological condition is selected from the groupconsisting of osteoporosis, hypocalcaemia of malignancy, multiplemyelomatosis, hyperparathyroidism, and hyperthyroidism. More preferably,the pathological condition is osteoporosis.

The polypeptides or the polynucleotides of the present inventionemployed in the methods described herein may be free in solution,affixed to a solid support, borne on a cell surface, or locatedintracellularly. To perform the methods it is feasible to immobilizeeither the polypeptide of the present invention or the compound tofacilitate separation of complexes from uncomplexed forms of thepolypeptide, as well as to accommodate automation of the assay.Interaction (e.g., binding of) of the polypeptide of the presentinvention with a compound can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and microcentrifuge tubes. In one embodiment, afusion protein can be provided with adds a domain that allows thepolypeptide to be bound to a matrix. For example, the polypeptide of thepresent invention can be “His” tagged, and subsequently adsorbed ontoNi-NTA microtitre plates, or ProtA fusions with the polypeptides of thepresent invention can be absorbed to IgG, which are then combined withthe cell lysates (e.g., (35)³-labelled) and the candidate compound, andthe mixture incubated under conditions favorable for complex formation(e.g., at physiological conditions for salt and pH). Followingincubation, the plates are washed to remove any unbound label, and thematrix is immobilized. The amount of radioactivity can be determineddirectly, or in the supernatant after dissociation of the complexes.Alternatively, the complexes can be dissociated from the matrix,separated by SDS-PAGE, and the level of the protein binding to theprotein of the present invention quantiated from the gel using standardelectrophoretic techniques.

Other techniques for immobilizing protein on matrices can also be usedin the method of identifying compounds. For example, either thatpolypeptide of the present invention or the compound can be immobilizedutilising conjugation of biotin and streptavidin. Biotinylated proteinmolecules of the present invention can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylatin kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavichin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the polypeptides of the presentinvention but which do not interfere with binding of the polypeptide tothe compound can be derivatized to the wells of the plate, and thepolypeptide of the present invention can be trapped is the wells byantibody conjugation. As described above, preparations of a labeledcandidate compound are incubated in the wells of the plate presentingthe polypeptide of the present invention, and the amount of complextrapped in the well can be quantitated.

The polynucleotides of the invention of SEQ ID NO: 1-77 have been shownto increase osteoblast differentiation.

Accordingly, another embodiment of the present invention relates to amethod for in vitro production of bone tissue, comprising the steps ofcontacting undifferentiated mammalian cells with a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID No: 1-77, preferably selected from the group consisting of SEQ ID No:69-77 for a time sufficient to differentiate the undifferentiated cellsinto osteoblasts, thereby producing a continuous bone matrix.

In a preferred embodiment, the method comprises the steps of:

-   -   (a) applying undifferentiated mammalian cells on a substrate to        form a cellular substrate,    -   (b) introducing a polynucleotide sequence or a vector comprising        a nucleotide sequence selected from the group consisting of SEQ        ID NO: 1-77, preferably selected from the group consisting of        69-77, for a time sufficient to differentiate the        undifferentiated cells into osteoblasts, thereby producing a        continuous bone matrix.

The invention thus provides a method for producing a substrate with amatrix grown thereon, which can be used for the provision of load-bearimplants, including joint prostheses, such, as artificial hip joints,knee joints and finger joints, and maxillofacial implants, such asdental implants. It can also be used for special surgery devices, suchas spacers, or bone filers, and for use in augmentation, obliteration orreconstitution of bone defects and damaged or lost bone. Bone formationcan be optimized by variation in mineralization, both by inductive andby conductive processes.

A combination of the provision of a load-bearing implant (preferablycoated with a matrix as described above) with a bone filler comprising amatrix as described, constitutes an advantageous method according to thepresent invention.

The method of the invention is also very suitable in relation torevision surgery, i.e., when previous surgical devices nave to bereplaced.

suitable undifferentiated cells are bone marrow cells, includinghematopoietic cells and in particular stromal cells. The marrow cells,and especially the stromal cells are found to be very effective in thebone producing process when taken from their original environment.

The undifferentiated cells can be directly applied on the substrate orthey can advantageously be multiplied in the absence of the substratebefore being applied on the substrate. In the latter mode, the cells arestill largely undifferentiated after, multiplication and, for thepurpose of the invention, they are still referred to asundifferentiated. Subsequently, the cells are allowed to differentiate.Differentiation can be induced or enhanced by the presence of suitableinductors, such as glucocorticoids, and dexamethasone. Especiallysuitable inductors of differentiation are the expression inhibitoryagents of the present invention.

The use of undifferentiated cells provides several advantages. Firstly,their lower differentiation implies a higher proliferation rate andallows the eventual functionality to be better directed and controlled.Moreover, culturing these cells not only produces the required bonematrix containing organic anal inorganic components, but also results inthe presence, in the culture medium and in the matrix, of severalfactors which are essential for growth of the tissue and for adaptationto existing living tissue. Also, the culture medium can be a source ofactive factors such as growth factors, to be used in connection with theimplanting process. Furthermore, such undifferentiated cells are oftenavailable in large quantities and more conveniently than e.g., maturebone cells, and exhibit a lower morbidity during recovery. Moreover, theundifferentiated cells can be obtained from the patient for whom theimplant is intention. The bone resulting from these cells is autologousto the patient and thus no immune response will be induced. Matrices asthick as 100 μm can be produced as a result of the use ofundifferentiated cells.

The substrate on which the undifferentiated cells can be applied andcultured can be a metal, such as titanium, cobalt/chromium alloy orstainless steel, a bioactive surface such as a calcium phosphate,polymer surfaces such as polyethylene, and the like. Although lesspreferred, siliceous material such as glass ceramics, can also be usedas a substrate. Most preferred are metals, such as titanium, and calciumphosphates, even though calcium phosphate as not an indispensablecomponent of the substrate. The substrate may be porous or non-porous.The cells can be applied at a rate of e.g., 10³-10⁶ per cm², inparticular 10⁴-2×10⁵ cells per cm².

The culture medium to be used in the method according to the inventioncan be a commonly known culture medium such as MEM (minimum essentialmedium). Advantageously, the medium can be a conditioned medium. In thiscontext, a conditioned medium is understood to be a medium whereinsimilar cells have previously been incubated, causing the medium tocontain factors such as polypeptides, secreted by the cells which areimportant for cell growth and cell differentiation.

The cells are cultured for a time sufficient to produce a matrix layer,e.g., a matrix layer having a thickness of at least 0.5 μm, inparticular from 1 up to 100 μm, more in particular of 10-50 μm. Thecells may be contacted with the culture medium for e.g. 2-15 weeks, inparticular 4-10 weeks.

The production of the matrix, when applied on a substrate, results in acontinuous or quasi-continuous coating covering the substrate for atleast 50%, in particular at least 80% of its surface area.

The present invention further relates to the osteoblast cells obtainableby the above method.

The invention is further illustrated in the following figures andexamples.

FIG. 1. Intramenbranous and endochondral ossification,

FIG. 2. Principle of the osteoblast differentiation assay.

FIG. 3. Lay-out of the 96 well knock-down control plate.

FIG. 4. Lay-out of the 384 well control plate.

FIG. 5. Performance of the knock-down control plate in the AP assay

FIG. 6. Dot plot representation of raw data for one SilenceSelectscreening plate

FIG. 7. Analysing the upregulation of BAP-mRNA versus PLAP- or IAP-mRNA

FIG. 8. Results mineralization assay

FIG. 9. Pipetting scheme used for screening the Ad-shRNAs at MOIs.

EXAMPLES Example 1 Development of a High-Throughout Screening Method forthe Detection of Endogenous Alkaline Phosphatase

Principle of the Assay

Mesenchymal progenitor cells (MPCs) are determined to differentiate intoosteoblasts in the presence of appropriate factors (e.g. BMP2). An assayto screen for such factors was developed by monitoring the activity ofalkaline phosphatase (AP) enzyme, and early marker in the osteoblastdifferentiation program. MPCs were seeded in 384 well plates andsimultaneously co-infected one day later with adenoviruses encoding thehuman coxsackie and adenovirus receptor (hCAR; Ad-hCAR) and individualsiRNA adenoviruses (Ad-siRNA) from the SilenceSelect™ collection.AdC15-hCAR/AdC20-hCAR co-infection increases the AdC01-siRNA infractionefficiency. Cellular AP activity was determined 13 days after the startof the infection (13 dpi). FIG. 2 illustrates the principle of theassay.

Development of the Assay

MPCs were isolated from bone marrow of healthy volunteers, obtainedafter informed consent (Cambrex/Biowhittaker, Verviers, Belgium).

In a series of experiments, carried out in 384 well plates, severalparameters were optimized: cell seeding density, multiplicities ofinfection (MOI) of control viruses (Ad-BMP2 or Ad-eGFP), MOI of Ad-hCAR,duration of infection, toxicity, infection efficiency (using Ad-eGFP)and the day of readout.

Using Ad-BMP2 (BMP2 overexpression) as a positive control for assaydevelopment, the following protocol resulted in the highest dynamicrange for the assay with the lowest standard deviation on the backgroundsignal: MPCs were seeded on day0 at 500 cells per well of a 384 wellplate and co-infected the next day using a mix of Ad-hCAR (5 μl of anAd-hCAR solution: mix total MOI=155.7) and 1 μl of Ad-control-virus(Ad-BMP2 or Ad-eGFP; corresponds to a theoretical MOI of 5000). On day5,the medium containing virus was removed and replaced by fresh mediumcontaining no virus. Upregulation of alkaline phosphatase was read at 13dpi: 15 μl 4-Methylumbelliferylphosphate (MUP, Sigma) was added to eachwell, the plates were incubated for 15 min at 37° C. and monitored forAP activity using a fluorescence plate reader (Fluostar, BMG).

After optimisation of the assay, a small pilot screen was run (103different Ad-siRNA viruses) with the use of robotics (96/384 channeldispenser Tecan Freedom 200 equipped with TeMO06, TeMO384 and RoMa,Tecan AG, Switzerland). The hits from this screen were collected andretested in the same assay. The two Ad-siRNAs that scored strongest(H9=H24-010; H10=H24-011) were used to generate a control plate(knock-down (KD) control plate) containing Ad-siRNAs. The control plate,a 96 well plate containing 3 negative (N1,N2,N3) and 3 positive(P1,P2,P3) control viruses is depicted in FIG. 3. This “knock-down”control plate contains Ad-H9 (h24-010) and Ad-H10 (H24-011) as positivecontrols; Ad-eGFP (knock-in virus) as infection control; andAd-eGFP-siRNA, Ad-M6PR-siRNA and Ad-Luc-siRNA (all 3 are knock-downviruses) as negative controls.

The control viruses were pipetted from 96 well KD control plates into384 well plates using robotics. The final lay-out of the 384 well plateis depicted in FIG. 4.

FIG. 5 shows results from the automated screening procedure using the KDcontrol plate. The mean and standard deviations of the KD negativecontrols (N1-N3) were used to calculate a cut-off for hit analysis,which was set at the mean for N1, N2, N3 (‘All negatives’) plus 3 timesthe standard deviation for ‘All negatives’. The positive controls (P1and P2), scored in more than 95% of the infected wells. The negativecontrol viruses scored in less than 5% of the wells.

Example 2 Screening of 2760 Ad-siRNA Adenoviruses in the OsteogenesisAssay

The optimized protocol for screening the SilenceSelect library is thefollowing: on day 0, MPC cells are seeded in black 384 well plates withclear bottom (Costar or Nunc) in 60 μl medium at a density of 500 cellsper well. One day later, 1 μl Ad-siRNA virus form the SilenceSelect™collection, stored in 384 cell plates (estimated titer of 2.5×10⁹ viralparticals per ml) and 5 μl of Ad-hCAR solution (total MOI=155),dispensed in 96 well V-bottom plates, is transferred with the aid of a96/384 channel dispenser (Tecan Freedom 200 equipped with TeMO96,TeMO384 and RoMa, Tecan AG, Switzerland) from the wells of a 96 wellplate containing the Ad-hCAR solution to each of the wells of the 384well plates containing MPCs. The KD control plate was run under the sameconditions as the aliquot plates from the SilenceSelect collection. AllAd-siRNA viruses were screened in duplicate, with each singular on adifferent MPC plate. Plates were then incubated at 37° C. Four days postinfection the medium containing the adenoviruses was replaced by freshmedium free of virus. Thirteen days post infection, the AP activityreadout was performed. A typical result of a 384 well screening plate isdepicted in FIG. 6, in which the relative fluorescence units (RFU) areplotted for each of the data points of the 384 well plate on the Y-axis;while the numbers on the X-axis correspond to positions in the 384 wellplate.

This duplicate screen was done twice, and all four data points were usedfor hit calling (see Example 3).

Example 3 Target Identification Using the AP Assay

After performing these 2 screens, the data obtained from measuring theAP activity were analyzed as follows: the background was calculated bytaking the mean of the data points from all the plates except thecontrol plate. A cut-off value for hit calling was calculated by adding3 times the standard deviation of all data points, excluding the controlplate. Each data point was analyzed for scoring above or under thecut-off. Only Ad-siRNAs including endogenous AP activity levels abovethe cut-off were of further interest. Hits were prioritized according totheir scoring in single or duple, in one or both of the screens. Datawere collected for 2688 Ad-siRNA virus constructs representing 2657independent KD constructs and are listed in table 2. One of theidentified hits has been shown to be a bone anabolic factor before andtherefore validates the assay:

H24-241: BMP3

BMP3 is a member of the bone morphogenetic protein family of secretedproteins. BMP3 functions as a antagonist for the osteogenic BMP2.BMP3-null mice have twice as much trabecular bone as wild-type animalsindicating that BMP3 is a negative regulator of bone homeostasis in vivo(Daluiski et al., Nature Genetics (2001) 27:84-88).

Example 4 Quality Control of the Target Ad-siRNAs

The Ad-siRNA hits were subjected to a quality control on the siRNAinsert.

Target Ad-siRNAs were propagated using PerC6 cells (Crucell, Leiden, TheNetherlands) at a 96 well plate level, followed by rescreening theseviruses at several MOIs in the primary assay (see Example 1) and bysequencing the siRNAs encoded by the target Ad-siRNA viruses.

PerC6/E2A cells were seeded in 96 well plates at a density of 40 000cells per well in 180 μl PerC6/E2A medium. Cells were then incubatedovernight at 39° C. in a 10% C0₂ humidified incubator. One day later,cells were infected with 1 μl of crude cell lysate from SilenceSelectstocks containing target Ad-siRNAs. Cells were incubated further at 34°C., 10% CO₂ until appearance of cytopathic effect (as revealed by theswelling and rounding up the cells, typically 7 days post infection).The supernatant was collected and the virus crude lysate was treatedwith proteinase K: 12 μl crude lysate was added to 4 μl Lysis buffer (1×Expand High Fidelity buffer with MgCl₂ (Roche Molecular biochemicals,Cat. No 1332465) supplemented with 1 mg/ml proteinase K. (RocheMolecular Biochemicals, Cat No 745 723) and 0.45% Tween-20 (RocheMolecular Biochemicals, Cat No 1335465) in sterile PCR tubes. These wereincubated at 55° C. for 2 h followed by a 15 min inactivation step at95° C. For the PCR reaction, 1 μl lysate was added to a PCR master mixcomposed of 5 μl 10× Expand High Fidelity buffer with MgCl₂, 0.5 μl ofdNTP mix (10 mM for each dNTP), 1 μl of ‘Forward primer’ (10 mM stock,sequence: 5′ CCG TTT ACG TGG AGA CTC GCC, SEQ ID NO: 311), 1 μl of‘Reverse Primer’ (10 mM stock, sequence: 5′ CCC CCA CCT TAT ATA TAT TCTTTC C, SEQ ID NO: 312), 0.2 μl of Expand High Fidelity DNA polymerase(3.5 U/μl, Roche Molecular Biochemicals) and 41.3 μl of H₂O. PCR wasperformed in a PE Biosystems GeneAmp PCR system 9700 as follows: the PCRmixture (50 μl in total) was incubated at 95° C. for 5 min; each of 35subsequent cycles ran at 95° C. for 15 sec, 55° C. for 30 sec, 68° C.for 4 min. A final incubation at 68° C. was performed for 7 min. 5 μl ofthe PCR mixture was mixed with 2 μl of 6× gel loading buffer, loaded ona 0.8% agarose gel containing 0.5 μg/μl ethidium bromide to resolve theamplification products. The size of the amplified fragments wasestimated from a standard DNA ladder loaded on the same gel. Theexpected size was ˜500 bp.

For sequencing analysis, the siRNA constructs expressed by the targetadenoviruses were amplified by PCR using primers complementary to vectorsequences flanking the SapI site of the pIPspAdapt6-U6 plamid. Thesequence of the PCR fragments was determined and compared with theexpected sequences.

Example 5 Analysis of the Upregulation of Endogenous Bond AP mRNA Versusthat of Placental or Intestinal AP mRNA

BAP is the physiologically relevant AP involved in bone formation. Inorder to determine whether the measured AP activities were due toupregulation of BAP expression or of another AP gene product, mRNAlevels for all AP genes were analysed for infected MPCs.

mRNA levels were determined as described in the previous sections. Thedifference is in the primer set used (see Table 1): one set detects BAPALPL (human alkaline phosphatase liver/bone/kidney) mRNA expression.Another set detects the expression of the 3 other AP genes (ALPL (humanalkaline phosphatase intestinal), ALPP (human alkaline phosphataseplacental (PLAP)), and ALPPL2 (human alkaline phosphataseplacental-like)). ALPI, ALPP and ALPPL2 are highly similar at theneculeotide level and can therefore be amplified using one primer pair.

The primer pairs were first validated on RNA isolated from MPCs infectedwith Ad-eGFP and Ad-BMP2. FIG. 7 illustrates the strong upregulation ofBAP mRNA by Ad-BMP2 and the absence of upregulation of expression of anyof the other AP genes. MPCs were infected in 24 well plate format usingAd-eGFP (negative control) or the osteogenic Ad-BMP2. Cells wereharvested and RNA was prepared and subjected to rtRT-PCR using primersets amplifying BAP mRNA or mRNA from the other 3 AP genes (PLAP/IAP).Ad-BMP2 strongly upregulates BAP mRNA levels but not the mRNA levels ofthe other 3 AP genes.

Both primer sets were then used to measure mRNA levels for all AP genesin RNA isolated from Ad-siRNA infected MPCs.

TABLE 1 AP primer sets SEQ ID Name Sequence NO: JDO-05F (PLAP)TTCCAGACCATTGGCTTGAGT 313 JDO-05bis R ACTCCCACTGACTTTCCTGCT 314(PLAP/ALPI/ALPPL2) JDO-21F (BAP) CATGCTGAGTGACACAGACAAGAAG 315JDO-21R (BAP) TGGTAGTTGTTGTGAGCATAGTCCA 316

Example 6 Mineralization

The process of osteogenesis consists of several successive events.During the initial phases of osteogenesis, bond alkaline phosphatase(BAP) becomes upregulated. It is however equally important to look atspecific events occurring in later stages of osteogenesis such asmineralization.

Assay Setup

The process of osteogenesis consists of several successive events.During the initial phases of osteogenesis, bone alkaline phosphatase(BAP) becomes upregulated. Later, during differentiation, cells deposit(hydroxy)apatite (Ca²⁺-phosphate precipitate) on an extracellular matrixconsisting mostly of collagen type I to form mineralized bone.

In the bone cell mineralizing assay (BM assay), primary human MSCs aredifferentiated in vitro into mineralizing osteoblasts using BMP2(recombinant or delivered by adenoviral transduction) as an osteogenicagent. Mineralization is then visualized by staining the MSCs withAlizarin Red, a dye with a high affinity for calcium (see FIG. 8).

Screening and Hit Calling

The following optimized protocol was used for screening Ad-siRNA andAd-cDNA targets identified in the primary assay: 100,000 MPCs wereseeded in each well or a 6 well plate in 2 ml MSC medium, containing 10%FCS. The neat day, after incubation at 37° C., 10% CO₂ in a humidifiedincubator, calls were co-infected with adC15-hCAR (final MOI of 750) andAd-siRNA, Ad-cDNA or control viruses as a final MOI of 1250, 2500 and5000. Cells were incubated at 37° C., 10% CO₂ in a humidified incubatorfor a further six days. Virus was removed and replaced by 2 ml fresh MSCmedium, 10% FCS. Over the next 22 days, medium was refreshed 3 times in2 weeks. Every other time, medium was refreshed half or completely. At28 days after the start of the experiment, the conditioned medium wasremoved, cells were fixed using 10% paraformaldehyde and the monolayersstained with 1 mL of ˜1% Alizarin Red (Sigma, #A5533) in MilliQ water(pH adjusted to 4.2). Ad-eGFP, to assets infection efficiently, Ad-BMP2as strong osteogenic inducer and Ad-H4-2 as a weak osteogenic factorwere included in each experiment as negative and positive controls,respectively. Every experiment where Ad-H4-2 did not inducemineralization was entirely repeated.

The Ad-shRNAs that induced mineralization are presented in Table 2.

Example 7 Drug Discovery Against the Identified Targets

Compounds are screened for binding to the polypeptides of the presentinvention. The affinity of the compounds to the polypeptides isdetermined in a emplacement experiment. Such displacement experimentsare well known in the art, and can be considered as a common techniqueamong others to identify compounds that bind to polypeptides.

In brief, the polypeptides of the present invention are incubated with alabeled (radio-labeled, fluorescent- or antibody-labeled, or any otherdetectable label) ligand that is known to bind to the polypeptide and isfurther incubated with an unlabeled compound.

The displacement of the labeled ligand from the polypeptide isdetermined by measuring the amount of labeled ligand that is stillassociated with the polypeptide. The amount of the labeled ligandassociated with the peptide is an indication of the affinity for theunlabeled compound.

The amount of labeled ligand associated with the polypeptide is plottedagainst the concentration of the unlabeled compound to calculate IC50values. The value reflects the binding affinity of the unlabeledcompound to its target, i.e. the polypeptides of the present invention.

Compounds are considered strong binders, when having an IC50 in thenanomolar and even picomolar range. Compounds that have an IC50 of atleast 10 micromol or even better in the nmol to pmol range are appliedin either the bone alkaline phosphatase assay (BAP) and/or in assays todetermine their effect on the induction of osteoblast markers andosteoblast function. Compounds with a lower IC50 are generallyconsidered as of less interest. The polypeptides of het presentinvention can be prepared in a number of ways depending on whether theassay will be run on cells, cell fractions or biochemically, on purifiedproteins. Such preparations are well known in the art, as are thedifferent assays.

Example 8 Osteoclast Assays: Validate Anti-Resorptive Activity ofIdentified Targets

Throughout life, the skeleton is in a constant state of remodeling.Focal areas of bone are resorbed by osteoclasts and then replaced bybone matrix newly formed by osteoblasts. The development of osteoporosisis characterized by severe bone loss due to the deregulation of thebalance between osteoclast and osteoblast activity, leading to anincreased osteoclast-mediated bone resorption.

Osteoclasts emanate from cells of the monocyte/macrophage lineage. Invivo, the differentiation of osteoclast precursor cells towardsosteoclasts is controlled by two central factors expressed by stromalcells (MPCs): receptor activator of NF B ligand (RANKL) andosteoprotegerin (OPG). RANKL is a membrane bound ligand expressed on thesurface of MPCs which drives osteoclast differentiation. OPG is asoluble decoy receptor for RANKL which inhibits osteoclastdifferentiation by scavenging active RANKL. The balance between RANKLand OPG expression by MPCs determines the level of osteoclastdifferentiation.

As MPCs control the differentiation of osteoclasts, it is important toknow the effect of the identified target Ad-siRNAs on osteoclastdifferentiation or activity. Target Ad-siRNAs that decrease osteoclastdifferentiation/activity, are very valuable, as these are expected toincrease bone apposition by two mechanisms: increase ofdifferentiation/activity of osteoblasts and decrease in osteoclastactivity. As illustrated by various precedents (Thirunavukkarasu et al.,(2000) J Biol Chem 275: 25163-72; Yamada et al., (2003) Blood 101:2227-34) such a pleiotropic effect of osteogenic factors can beexpected.

Osteoclast Differentiation Assay

The effect of osteogenic factors on osteoclastogenesis is evaluatedthrough two types of assays.

In a first assay setup, a coculture of MPCs with primary humanmonouclear cells is performed. The effect of the infection of the MPCmonolayer with a knock-down virus on its capacity to supportosteoclastogenesis is evaluated. The desired effect is the following:knock-down of the Ad-siRNA target gene expression in the MPCs shouldinhibit osteoclast differentiation driven by a physiological trigger ase.g. a mixture of 10 nM 1.25(OH)₂vitD₃ and 50 nM M-CSF. The monocytesused can be derived from bone marrow or peripheral blood. In the presentexample, a differentiation experiment based on peripheral blood derivedmononuclear cells (PBMCs) is described. MPCs (obtained fromCambrex/Biowhittaker, Vervieers, Belgium) are seeded in 96 well plates(1000 cells per well) in -MEM medium (GIBCO-Life Technologies)supplemented with 10% FBS and a day later, these are infected with atarget Ad-siRNA. At least three days later, 100 000 PBMCs per well areadded as well as M-CSF (R&D systems, 50 ng/ml final concentration). Halfthe volume of medium is refreshed twice a week by medium+50 ng/ml M-CSFand 10 nM 1.25(OH)₂vitD₃. Readout is performed 14 days after addition ofthe PBMCs to the coculture. Spontaneous osteoclast differentiationdriven by the multiple readouts. Microscopic assessment of the number of‘TRAP positive’, multinucleated cells per well is a generally acceptedmeasure for the level of osteoclast differentiation. ‘TRAP positive’means that the cells possess a tartrate resistant acidic phosphatase(TRAP) activity. To assess this, the coculture is subjected to an insitu TRAP straining performed according to the Acid Phosphatasedetection kit (SIGMA, 386-A). Positive cells acquire a purple color upontreatment. As an alternative readout, a marker specific for matureosteoclasts is measured e.g. TRACP5b (tartrate resistant acidicphosphatase type 5b), calcitonin receptor (CTR) or Cathepsin K (CTSK).Measurement of the amounts of osteoclast-derived tartrate resistantacidic phosphatase protein (TRACP5b) in the coculture supernatant isperformed by a commercially available ELISA (BoneTRAP assay, Sbasciences, Turku, Finland). CTR or CTSK are detected byimmunocytochemistry, upon application of following general protocol.Medium is removed and the coculture is fixed (4% paraformaldehyde, 0.1%TritonX-100, 4° C., 30 min), washed and blocking buffer (PBS+1% BSA+0.1%Tween20) is added for an incubation of at least 4 hrs. The blockingbuffer is removed and the primary antibody directed against CathepsinK(e.g. Oncogene, IM55L) or Calcitionin receptor (e.g. Serotec, AHP635),dissolved at the desired concentration in a suited buffer (e.g. 0.05MTris.HCl pH 7.4, 1% BSA), is added to the wells. Incubation is performedovernight, 4° C. The mixture is removed, the cells washed (PBS+0.1%Tween20) and the suited, HRP conjugated secondary antibody, diluted inthe same buffer as the primary antibody, is added. After an incubationof at least 4 hrs, a washing step is performed (PBS+0.1% Tween20) andluminol (a substrate for HRP yielding a luminescent signal: BMChemiluminescence ELISA Substrate [POD] (luminol), Roche Diagnostics,Cat No 1582950) is added. After 5 min incubation, readout is performedwith a luminometer (Luminoskan Ascent, Labsystem). The 2 assaysdescribed (assessment of the amount of multinuclear cells andimmunochemistry for the detection of osteoclast-specific markers) allowto assess the differentiation of the mononuclear cells towardsosteoclasts, but do not yield information about the bone resorptiveactivity of the osteoclasts formed.

Activity of the osteoclasts is measured in the pit formation assay. Forthis purpose, the co-culture and infection of cells is performed asdescribed for assays described above with the difference that abone-like substrate is present at the bottom of the well in which theco-culture is performed. This bone-like substrate can be a dentin slice(e.g. Kamiya Biomedical Company, Seattle (Cat No KT108)) or equivalent(Calcium carbonate coating, OAAS™, Gentaur; Biocoat™ Osteologic™, BDBiosciences) that is commercially available. The co-culture is performedfor at least 14 days on the bone like substrate. Cells are then removedby treatment with sodium hypochlorite and the area resorbed by theosteoclasts (the resorption pit) can be assessed microsopically. Thiscan be facilitated by the treatment of the surface of the dentin slicewith toluidine blue.

In a second assay setup, the effect of the infection of the osteoclastprecursor cells (PBMCs or BMMCs) with a hit virus on its ability todifferentiate towards an osteoclast is measured in a monoculture assay.For this purpose, the monocytes (PBMCs or BMMCs) are seeded in a 384well plate in MEM medium supplemented with 10% serum and 25 ng/mlrecombinant, M-CSF (R&D systems). One day after seeding, the cells areinfected with target Ad-siRNAs. Four days after infection, recombinantRANKL is added to the wells (25 ng/ml, R&D systems). Medium is refreshedtwice a week. Fourteen days after addition of RANKL, the differentiationof the monocytes towards osteoclasts is measured using one of thereadouts described for the former assay setup. This assay allows theidentification of factors that are indispensable for the response ofosteoclast precursor cells to M-CSF or RANKL.

PBMC Isolation

PBMCs are obtained from peripheral blood (obtained from patients afterinformed consent) subjected to the following protocol. Blood isaseptically poured into 50 ml Falcon tubes and spun at 3000 g for 10 minat 25° C. The buffy coat is then collected and diluted 1:1 with PBS. Thediluted buffy coat is poured on top of 20 ml Lymphoprep (Sigma)contained in a 50 ml Falcon tube. Upon centrifugation (35 min at 400 gat 25° C.), a white layer of monomuclear cells on top of the Lymphorprepis collected and washed twice with PBS (centrifugation at 200 g, 10 min,25° C.) and rediluted in 7 ml PBS. This solution is pipetted onto alayer of 7 ml of hyperosmolar Percoll gradient contained in a 15 mlFalcon tube and centrifuged 35 min at 400 g at 25° C. The hyperosmolarPercoll gradient is prepared as follows; 1 volume of 1.5 M NaCl and 9volumes of Percoll (Pharmacia, d=1,130 g/ml) are mixed. This mixture isadded 1:1 to a PBS/Citrate buffer (NaH2PO4 1.49 mM, Na2HPO4 9.15 mM,NaCl 139.97 mM, Na-citrate (dihydrate) 13 mM, pH 7.2). Aftercentrifugation, monocytes form a discrete ring on top of the gradient.Monocytes are collected and washed in culture medium. Cells are thenready to use in assays.

Example 9 Analysis of ‘Off-Target’ Knock Down Effect

SiRNAs exert knock-down of gene expression through a recently discoveredand partially understood mechanism. It is generally accepted that thespecific annealing of the siRNA sequence to mRNA is responsible for agene-specific ‘on-target’ knock-down. However, it cannot be excluded yetthat limited mismatching between the siRNA and another mRNA can induce‘off-target’ down-regulation of gene expression. In order to excludethat the knock-down of (an) ‘off-target’ mRNA(s) was responsible for theobserved osteogenic effect, additional siRNAs/shRNAs were designed for 5targets (Table 2B) that induced mineralization using stringent designcriteria. The additional Ad-shRNAs were then tested in the BAP assay.

Galapagos has developed a proprietary algorithm that incorporates bothpublished and proprietary design criteria (The Galapagos algorithmsincorporates published criteria for siRNA design such as the Tuschlrules and rules from Reynolds et al. Nat. Biotechnol. 2004 Mar.22(3):326-30) The latter include criteria such as low thermodynamicinternal stability at the 5′ antisense end of the RNAi sequence ‘GCcontent’). To address the question of possible ‘off-target’ effects,additional siRNA sequences were designed that:

-   -   align perfectly with the mRNA targeted by the original siRNA    -   that may align imperfectly (maximum of 2 basepairs non-identity        checked for every position of the 19 mer) with a minimal number        of ‘off-target’ mRNAs such that        -   putative ‘off-target’ mRNAs were different from the putative            ‘off-target’ mRNAs identified for the original siRNA.        -   putative ‘off-target’ mRNAs were different from the            putataive ‘off-target’ mRNAs identified for all original            target siRNAs, except for the additional siRNAs designed for            PPIA

7 additional siRNAs, designed for each of the 5 selected target geneswere processed to derive recombinant adenoviruses. All siRNAs weresequenced upon cloning, to verify their identity and exclude errors dueto the oligonucleotide synthesis.

34 Ad-shRNAS were successfully generated and tested in the BAP assay at3 MOIs in 2 independent experiments, in parallel with the original 5Ad-shRNAs.

Recombinant adenoviruses encoding the designed shRNAs (Ad-shRNAs) wereproduced, titered, aliquoted in 96 well plates and stored at −80° C.These plates were processed in the primary BAP assay as follows:

MPC cells were seeded with a Multidrop 384 (Labsystems) in black 384well plates with clear bottom (Costar or Nunc) in 60 μl MSC mediumcontaining 10% fetal calf serum (FCS) (proprietary medium fromProgentix, The Netherlands), at a density of 500 cells per well. One daylater, a 96 cell plate containing aliquoted Ad-shRNAs and anothercontaining negative and positive control viruses (knock-down controlplate, FIG. 3) were thawed and virus aliquots transferred to the MPCplate using a 96-channel dispenser (Tecan Freedom 200 equipped with aTeMO96 and a RoMa plate handler, Tecan AG, Switzerland) (FIG. 9). Forthe control plate, 1 μL virus stock (average titer of of 2×10⁸ viralparticles per ml) was transferred to the 384 well screening plates. TheAd-shRNAs were screened at 3 multiplicities of infection (MOIs): 12,000,4,000 and 1,333. Viruses were transferred from the 96 well stock plateto three 384 well screening plates (FIG. 9). Next, 5 μl of adenovirusexpressing the human coxsackie and adenovirus receptor (hCAR)(AdC15-hCAR/AdC20-hCAR) was transferred into these wells (final MOI of155) from a 96 well V-bottom plate with the aid of the 96-channeldispenser.

Plates were then incubated at 37° C., 10% CO₂ in a humidified incubatorfor four days. Four days post infection, the medium containing theadenoviruses was replaced by 60 μl fresh MSC medium containing 10% FCSfree of virus. After an additional nine days of incubation, medium wasremoved, 15 μL of a 4-methylumbelliferylphosphate solution (Sigma,#M3168) was added to each well, and the fluorescence of4-methyl-umbelliferone released by the alkaline phosphatase activity wasmeasured after 15 min incubation at 37° C. using a fluorimeter(excitation; 360 nm; emission: 440 nm; FluoStar, BMG).

All Ad-shRNAs viruses were screened in duplicate at 3 MOIs in twoidentical but independent screens. Thresholds were calculated for hitcalling using either all negative controls present in one screeninground (‘Global’ analysis) or using the negative controls present on onescreening plate (‘Local’ analysis). Hits were called according to thefollowing selection criteria:

-   -   1) BAP signals higher than the mean plus 3 times (+3) the        standard deviation of negative controls. The two individual        datapoints for each virus in the batch were analyzed        independently.    -   2) Positive BAP signals as defined by criterion 1 where one        Ad-shRNAs scores at least at one MOI in duplicate in at least        one of the 2 screens.

A ‘Global’ analysis of the data identified 8 siRNAs targeting 5 loci anda ‘Local’ analysis’ identified 9 siRNAs targeting 5 loci. The identityof the 5 selected genes is presented in Table 3 together with the finalnumber of siRNAs that scored in the BAP assay. All original 5 Ad-shRNAsscored in the BAP assay based on both the ‘Global’ and ‘Local’ analysis.

TABLE 3 Identification of multiple siRNAs for selected validated targetsin the BAP assay. Global analysis - Local analysis - Gene ID RedundancyRedundancy AGTRL1 2 3 DRD5 3 3 ENSG00000172441 2 2 GPR39 3 3 PDE11A 3 3

In Table 3, the numbers indicate all siRNAs that scored in the BAPassay, including the original 5 siRNAs.

In conclusion, additional Ad-shRNAs targeting 5 selected targets weredesigned and constructed. Negative controls present on the controlplates were used per plate (‘Local’ analysis) or per batch of plates(‘Global’ analysis) to determine the cutoff for hit calling (+3),

-   -   the ‘Global’ analysis resulted in 8 viruses that scored positive        in the BAP assay, confirming 5 of the 5 validated targets    -   the ‘Local’ analysis resulted in 9 viruses that scored positive        in the BAP assay, confirming 5 of the 5 validated targets    -   All original 5 Ad-shRNA viruses scored in the BAP assay when        using either the ‘Global’ or the ‘Local’ analysis.

TABLE 2ALists the polypeptides, polynucleotides and knockdown constructs of thepresent invention. Score in Mineral- isation assay as described TargetSEQ ID NO in KD Target Gene GenBank KD Poly- Poly- Example Hit IDSequence Symbol Accession Name construct nucleotide peptide 6 H24-TGCAGGCCCT SLC7A NM_003045 Solute carrier    1  78 194 229 GCCATTGTC 1family 7  (cationic  amino acid  transporter, y⁺ system),  member 1 H24-GAAGATTACAG PDLIM7 NM_005451- MDZ and LIM   2 79-81 195-197 230 GCGAGATCNM_203350- domain 7(enigma) NM_203353 H24- CTCTGGAAGG RAD50 NM_005732RAD50 homolog    3  82 198 231 AGTCATTAC (S.    cerevisiae) (RAD50),transcript  variant 1 H24- CACTAAGGTG AGTRL NM_005161 angiotensin II   4 83 199 X 233 CAGTGCTAC 1 receptor-like 1 H24- GGTGTACTTCA SCN4ANM_000334 sodium channel,   5  84 200 235 CCAACGCC voltage-gated, type IV, alpha H24- GCTTCTGAAGA KCNJ1 NM_000220- potassium in-   6 85-89201-205 236 CCACAGTC NM_153764- wardly-rectify  NM_153765-ing channel,   NM_153766- subfamily NM_153767 J, member 1 H24-CTACCTGCTG ENTPD NM_001246- ectonucleoside   7 90-91 206-207 237GAGAACTTC 2 NM_203468 triphosphate diphospho- hydrolase 2 H24-TGGCACAGTG CLCA1 NM_001285 chloride    8  92 208 238 ATCGTGGAC channel,calcium acti- vated, family  member 1 H24- CCTGTTCAGAA CLCN6 NM_001286chloride chan-   9  93 209 239 CGATGGGC nel 6 (CLCN6),  transcriptvariant CIC-6a H24- TTGCGCCAGG BMP3 NM_001201 bone  10  94 210 X 241AGATACCTC morphogenetic protein 3 (osteogenic)  (BMP3) H24- CGCCTTCAAAABCA5 NM_018672- ATP-binding 11 95-96 211-212 242 GAGAAATTC NM_172232cassette,  sub-family A (ABC1),  member 5 H24- CTCTCTGTGGT SLC2ANM_207420 Solute carrier  12  97 213 243 CAACACGC 7 family 2(facilitated  glucose transporter), member 7 H24- GGAAGGGTAT GPD2NM_000408 glycerol-3- 13  98 214 244 CTGGAAGCC phosphate dehydrogenase 2 (mito- chondrial) H24- CAGCAGGAAG GALNT NM_054110 UDP-N-acetyl- 14  99215 X 245 GAGATTCAC L2 alpha-D- galactosamine  poly-peptideN-acetylgalac- tosaminyl- transferase- like 2 H24- GCACCTGCAC B3Gn-NM_138706 beta-1,3-N- 15 100 216 X 246 TTGCTCGAC T6 acetylgluco- saminyltransferase  protein (B3Gn-T6) H24- GTTGACTAATC LOC25 XM_054745similar to 16 101 217 247 CTCCTTCC 7478 Neurogenic locus notch homologprotein 1  precursor (Notch 1) (hN1) (Translocation- associated notchprotein TAN-1) H24- CATGGAGTGC LOC25 XM_172351 similar to 17 102 218 248TCTAGATCC 4325 peptidylprolyl isomerase A (cyclophilin A) H24-TCGAAGAGGT NETO1 NM_138966- neuropilin (NRP) 18 103-104 219-220 249GCCGACCAC NM_153181 and folloid  (TLL)-like 1 H24- TTGGACAAATC PPP3RNM_147180 protein  19 105 221 250 AGGGTCTC 2 phosphatase 3 (formerly 2B), regulatory  subunit B (19 kD),  beta isoform(calcineurin  B, type II) H24- TCCAGAGTACT INSRR NM_014215- inslulin re-20 106-107 222-223 251 TCAGCGCC XM_043563 ceptor related  receptor H24-CCAATTTGCCT GRP14 NM_032503 G protein- 21 108 224 252 GTAGTGCC 5 coupledreceptor 145 H24- GTGGAAGGCG CTSL NM_001912- cathepsin L 22 109-110225-226 253 ATGCACAAC NM_145918 H24- CTTGTGGACA LOC12 XM_060167similar to 23 111 227 254 GGCCAGATC 6767 arylacetamide deacetylase(esterase) H24- GCATGAGTTTC SMOC2 NM_022138 SPARC related 24 112 228 255TGACCAGC modular  calcium binding 2 H24- CTATTGTTCCA LOC15 XM_099028LOC159121 25 113 229 X 256 GTGGAGGC 9121 H24- GTTTAAGGCA PDE11 NM_016953phospho- 26 114 230 X 257 GCCAACATC A diesterase 11A H24- CTTAGTTTCCASTK19 NM_004197- Serine/threonine 27 115-116 231-232 259 GCAGGACCNM_032454 kinase 19 H24- GATCGGGTTC SLC9A NM_004174 solute carrier  28117 233 260 CACCTGTCC 3 family 9  (sodium/hydrogen exchanger), isoform 3 H24- TTTGTGGTGTG HTR3A NM_000869- 5-hydroxy- 29 118-119234-235 261 CATGGCTC NM_231621 tryptamine (serotonin)  receptor 3A H24-TGCCAGCACC SLC9A NM_003047 solute carrier  30 120 236 262 ATTCGAAGC 1family 9  (sodium/hydrogen exchanger),  isoform 1 (antiporter, Na⁺/H⁺, amiloride sensitive) H24- GTCCGAGAGC GPR39 NM_001508 G protein- 31 121237 X 263 GAAGAGAGC coupled receptor 39 H24- GCAGGTGAAG ADRB1 NM_000684adrenergic,  32 122 238 264 AAGATCGAC beta-1- receptor H24- TAAGATTGAAGEPHX1 NM_000120 epoxide  33 123 239 265 GGCTGGAC hydrolase 1, microsomal(xenobiotic) H24- GGGTGTGGTC PRKD1 NM_002742 protein  34 124 240 266TGAATTACC kinsae D1 H24- CATCTTGCATG KCNJ1 NM_021012 potassium  35 125241 267 AGATTGAC 2 inwardly- rectifying  channel, subfamily J, member 12 H24- GAGATCCGGG AFG3L NM_006796 AFG3 ATPase  36 126 242 268AGAGAAATC 2 family gene 3- like 2 (yeast) H24- GTGCCGGATG SLC12NM_005072 solute carier  37 127 243 269 CGCATCTTC A4 family 12(potassium/ chloride transporters), member 4 H24- TACCAGTATGG ENTPDNM_001249 ectonucleoside 38 128 244 270 TGGCAACC 5 triphosphatediphospho- hydrolase 5 H24- GGGCTTCGTTT KCNK4 NM_016611- potassium  39129-131 245-247 271 CTGCTCTC NM_033310- channel, subfamily K, NM_033311member 4 H24- GCAAGTTCACT MAP2K NM_002758- mitogen- 40 132-133 248-249 X272 ACAGCATC 6 NM_031988 activated protein kinase kinase 6 H24-CTGTGCAGCT TIE1 NM_005424 tyrosine kinase  41 134 250 273 GCAGGGAACwith immunoglo- bulin-like and EGF-like domains 1 H24- TTGGAACAGCT ABCC1NM_032583- ATP-binding 42 135-137 251-253 274 GGACCAGC 1 NM_033151-cassette,  sub-family NM_145186 C (CFTR/MRP), member 11 H24- CCAGGATGTAEPHB2 NM_004442- EPH receptor  43 138-139 254-255 275 ATCAATGCCNM_017449 B2 H24- ACTCTCCGAAA LOC13 XM_059896 similar to 44 140 256 276GCATGGCC 7057 hypothetical  protein FLJ10661 H24- CGGAATGCAA SLC39NM_152725 Solute carier  45 141 257 277 GGAGATTGC A12 family 39 (zinc transporter), member 12 H24- GCTTCCAAGG HS3ST NM_153612-Heparan sulphate 46 142-143 258-259 X 278 AGCAGGTTC 5- XM_167035(glucosamine)  LOC22 3-O-sulfotrans 2537 ferase 5/  similar to  heparan sulfate D- glucosaminyl  3-O-sulfotrans- ferase 1 precursor;  heparin-glucosamine  3-O-sulfo- transferase H24- ATATACATTTC LOC16 XM_095086-similar to NADH 47 144-145 260-261 279 ACCCTAGC 8415- XM_172596dehydrogenase LOC25 subunit 4L 6726 H24- TCCCGCACTT ENSG0 ENSG000001Annotated using 48 146 262 X 280 GCCGAAGTC 000017 72441 proprietary 2441algorithms H24- TGCAAACGGC FOLR1 NM_000802- folate receptor  49 147-152263-268 281 ATTTCATCC NM_016724- 1 (adult) NM_016725- NM_016729-NM_016730- NM_016731 H24- CATCTTGCTGA SLC26 NM_022911- solute carrier 50 153-155 269-271 282 ACCTGGAC A6 NM_134263- family NM_13442626, member 6 H24- ATTAATGACCT SLC15 NM_005073 solute carrier  51 156 272283 CACAGACC A1 family 15  (oligopeptide transporter), member 1 H24-TGTCCAGGGA ENSG0 ENSG000001 KIAA1639  52 157-159 273-275 284 TATTGTGTC00012 24860- protein 4860 XM_290923- KIAA16 SK601 39- Obscn H24-GTGTCATGAG KCNS3 NM_002252 potassium  53 160 276 285 CTACCTTACvoltage-gated  channel, delayed- rectifier, subfamily S, member 3 H24-TGTGCTCAAAT SCNN1 NM_001039 sodium channel, 54 151 277 286 GACACCTC Gnonvoltage- gated 1, gamma H24- GGGTAGTCAG VTN NM_000638 vitronectin  55162 276 287 TACTGGCGC (serum spread- ing factor, somatomedin B,complement S- protein) H24- ACGTTTGGATA LOC16 XM_089158 similar to  56163 279 286 AAGTTGGC 3812 ALCOHOL DEHYDROGENASE CLASS III CHI CHAIN(GLUTATHIONE- DEPENDENT FORMALDEHYDE DEHYDROGENASE) (FDH) H24-TGAGCAGTTG NRD1 NM_002525 nardilysin  57 164 280 289 AAGAAGACC(N-arginine  dibasic convertase) H24- ACAACCTGGC DRD5 NM_000798dopamine  58 165 281 X 290 CAACTGGAC receptor D5 H24- GATCCAAGAG RASSFNM_007182- Ras associ- 59 186-172 282-288 291 GCCCTGCAC 1 NM_170712-ation (RalGDS/ NM_170713- AF-6) domain  NM_170714- family 1 NM_170715-NM_170716- NM_170717 H24- CCCATACTGTG ZNF354 NM_005649 Zinc finger  60173 289 292 GAGAAATC A protein 354A H24- GCGACTGGTG OLIG2 NM_005806oligodendrocyte 61 174 290 293 AGCGAGATC lineage  transcription factor 2H24- TGGTTCTCTTC PIK3R4 NM_014602 Phosphoino- 62 176 291 294 CCGACTGCsitide3-kinase,  regulatory subunit   4, p150 H24- AATCTTGTGCC PCM1NM_006197 pericentriolar 63 176 292 295 AAAGAGGC material 1 H24-ATGCCAGACA LOC38 XM_372233- Selenophosphate 64 177-178 292-294 296ATGCAGTGC 9873- NM_012247 synthetase 1 SEPHS 1 H24- GAGCAGACAA KCND2NM_012281 potassium  65 179 295 297 ACGAAGGGC voltage-gated channel, Shal- related  subfamily, member 2 H24- CTGTTCAGCA TNFRSNM_003790- tumor necrosis  66 180-189 296-305 X 296 GTGGCCGAC F25NM_148965- factor NM_148966- receptor NM_148967- superfamily, NM_148968-member 25 NM_148969- NM_148971- NM_148972- NM_148973- NM_148974 H24-ATGGCACTGT USP44 NM_032147 Ubiquitin  67 190 306 299 GTGGACTGC specificprotease 44 H24- GATCTCCACTG PCTK1 NM_006201- PCTAIRE protein 68 191-193307-309 300 AGGACATC NM_033018- kinase 1 NM_033019

TABLE 2B Addition to the summary of polynucleotides, polypeptides andknock-down constructs of the invention.

The invention claimed is:
 1. Method for identifying a compound thatinduces differentiation of undifferentiated mammalian cells intoosteoblasts, comprising (a) contacting a compound with isolatedundifferentiated mammalian cells overexpressing a polypeptide comprisingan amino acid sequence of SEQ ID NO: 237; and (b) measuring in saidmammalian cells the activation of a biological pathway producing abiochemical marker indicative of the differentiation of saidundifferentiated mammalian cells into osteoblasts.
 2. The method ofclaim 1, wherein said biological marker is bone alkaline phosphatase. 3.The method according to claim 1, wherein said compound is selected fromthe group consisting of compounds of a commercially available screeninglibrary and compounds having binding affinity for a polypeptidecomprising an amino acid sequence of SEQ ID NO:
 237. 4. The methodaccording to claim 3, wherein said compound is a peptide in a phagedisplay library or an antibody fragment library.
 5. A method foridentifying a compound that induces differentiation of undifferentiatedmammalian cells into osteoblasts, comprising (a) contacting a compoundwith a polypeptide consisting of an amino acid sequence of SEQ ID NO:237, in an in vitro cell-free preparation; (b) measuring the bindingaffinity of said compound to said polypeptide; and (c) selecting acompound for confirmation as an inducer of mammalian celldifferentiation into osteoblasts, which compound is selected based onits binding affinity for the polypeptide of SEQ ID NO.
 237. 6. A methodaccording to claim 5 for identifying a compound that inducesdifferentiation of undifferentiated mammalian cells into osteoblasts,said method further comprising (d) contacting said compound selected tohave binding affinity to said polypeptide of SEQ ID NO: 237 with anundifferentiated mammalian cell, which is in culture, and in which saidpolypeptide comprising the amino acid sequence of SEQ ID NO: 237 isexpressed; and (e) measuring in said culture at least one biochemicalmarker indicative of the differentiation of said undifferentiatedmammalian vertebrate cells into osteoblasts; and (f) determining if saidone or more biochemical marker indicative of the differentiation of saidundifferentiated mammalian vertebrate cells is upregulated as comparedto said biochemical marker expressed in said undifferentiated mammalianvertebrate cell that is not contacted with said compound; and (g)selecting a compound, based on its upregulation of the said biochemicalmarker, for confirmation as an inducer of mammalian cell differentiationinto osteoblasts.
 7. The method according to claim 6, wherein saidbiochemical marker is bone alkaline phosphatase, type-1 collagen,osteocalcin or osteopontin.
 8. The method according to claim 5 whereinsaid compound having binding affinity to said polypeptide of SEQ ID NO:237 exhibits a binding affinity of at least 10 micromolar.
 9. The methodaccording to claim 6, wherein said undifferentiated mammalian vertebratecell is an osteoblast progenitor cell.
 10. A method for identifying acompound that induces differentiation of undifferentiated mammaliancells into osteoblasts, comprising (a) contacting a compound with anundifferentiated mammalian cell in culture, in which cell has beenintroduced an expressible nucleic acid coding for and expressing a GPR39polypeptide comprising an amino acid sequence of SEQ ID NO: 237; (b)measuring a biochemical marker in the biological pathway of saidmammalian cell, in which biological pathway bone alkaline phosphatase isexpressed, which biochemical marker is expressed by saidundifferentiated mammalian cell, and which biochemical marker isindicative of the differentiation of said undifferentiated mammaliancells into said osteoblasts: (c) determining if said biochemical markerexpressed by said undifferentiated mammalian cells contacted with saidcompound measured in step (b) is upregulated as compared to saidbiochemical marker expressed in said undifferentiated mammalian cellthat is not contacted with said compound; (d) selecting a compound,determined to have upregulated said biochemical marker expressed by saidundifferentiated mammalian cells contacted with said compound, forconfirmation as an inducer of mammalian cell differentiation intoosteoblasts; (e) contacting said compound selected in step (d) with saidGPR39 polypeptide in an in vitro cell-free preparation; and (f)measuring the binding affinity of said compound to said GPR39polypeptide.
 11. The method according to claim 10, wherein saidundifferentiated mammalian cell is an osteoblast progenitor cell. 12.The method of claim 10 wherein said biochemical marker is bone alkalinephosphatase, type-1 collagen, osteocalcin or osteopontin.
 13. The methodaccording to claim 10, wherein said compound measured in step (f)exhibits binding affinity of at least 10 micromolar to said GPR39polypeptide.
 14. Method for identifying a compound that inducesdifferentiation of undifferentiated mammalian cells into osteoblasts,comprising (a) contacting a compound with an undifferentiated mammaliancell in culture, in which cell has been introduced an expressiblenucleic acid coding for and expressing a GPR39 polypeptide comprising anamino acid sequence of SEQ ID NO: 237; and (b) measuring in saidmammalian cells the activation of a biological pathway producing abiochemical marker indicative of the differentiation of saidundifferentiated mammalian cells into osteoblasts.
 15. The methodaccording to claim 14, wherein said undifferentiated mammalian cell isan osteoblast progenitor cell.
 16. The method of claim 14, wherein saidbiological marker is bone alkaline phosphatase.
 17. A method foridentifying a compound that induces differentiation of undifferentiatedmammalian cells into osteoblasts, comprising (a) contacting a compoundwith isolated undifferentiated mammalian cells overexpressing apolypeptide comprising an amino acid sequence of SEQ ID NO: 237; (b)measuring a biochemical marker in the biological pathway of saidmammalian cell, in which biological pathway bone alkaline phosphatase isexpressed, which biochemical marker is expressed by saidundifferentiated mammalian cell, and which biochemical marker isindicative of the differentiation of said undifferentiated mammaliancells into said osteoblasts: (c) determining if said biochemical markerexpressed by said undifferentiated mammalian cells contacted with saidcompound measured in step (b) is upregulated as compared to saidbiochemical marker expressed in said undifferentiated mammalian cellthat is not contacted with said compound; (d) selecting a compound,determined to have upregulated said biochemical marker expressed by saidundifferentiated mammalian cells contacted with said compound, forconfirmation as an inducer of mammalian cell differentiation intoosteoblasts; (e) contacting said compound selected in step (d) with saidpolypeptide comprising an amino acid sequence of SEQ ID NO: 237 in an invitro cell-free preparation; and (f) measuring the binding affinity ofsaid compound to said polypeptide comprising an amino acid sequence ofSEQ ID NO:
 237. 18. A method for identifying a compound that inducesdifferentiation of undifferentiated mammalian cells into osteoblasts,comprising (a) contacting a compound with an undifferentiated mammaliancell, which is in culture, and in which said polypeptide comprising theamino acid sequence of SEQ ID NO: 237 is expressed; (b) measuring abiochemical marker in the biological pathway of said undifferentiatedmammalian cell, which has been contacted with said compound, in whichundifferentiated mammalian cell biological pathway bone alkalinephosphatase is expressed, which biochemical marker is expressed by saidundifferentiated mammalian cell, and which biochemical marker isindicative of the differentiation of said undifferentiated mammaliancells into said osteoblasts; (c) determining if said biochemical markerexpressed by said undifferentiated mammalian cells contacted with saidcompound measured in step (b) is upregulated as compared to saidbiochemical marker expressed in said undifferentiated mammalian cellthat is not contacted with said compound; (d) selecting a compound,determined to have upregulated said biochemical marker expressed by saidundifferentiated mammalian cells contacted with said compound, forconfirmation as an inducer of mammalian cell differentiation intoosteoblasts; (e) contacting said compound selected in step (d) with saidpolypeptide comprising an amino acid sequence of SEQ ID NO: 237 in an invitro cell-free preparation; and (f) measuring the binding affinity ofsaid compound to said polypeptide comprising an amino acid sequence ofSEQ ID NO:
 237. 19. The method according to claim 17, wherein saidundifferentiated mammalian cell is an osteoblast progenitor cell. 20.The method according to claim 18, wherein said undifferentiatedmammalian cell is an osteoblast progenitor cell.
 21. The method of claim17, wherein said biochemical marker is bone alkaline phosphatase, type-1collagen, osteocalcin or osteopontin.
 22. The method of claim 18,wherein said biochemical marker is bone alkaline phosphatase, type-1collagen, osteocalcin or osteopontin.
 23. The method of claim 17,wherein said biological marker is bone alkaline phosphatase.
 24. Themethod of claim 18, wherein said biological marker is bone alkalinephosphatase.
 25. The method according to claim 17, wherein said compoundmeasured in step (f) exhibits binding affinity of at least 10 micromolarto said polypeptide comprising an amino acid sequence of SEQ ID NO: 237.26. The method according to claim 18, wherein said compound measured instep (f) exhibits binding affinity of at least 10 micromolar to saidpolypeptide comprising an amino acid sequence of SEQ ID NO:
 237. 27. Themethod of claim 1, wherein said marker is bone alkaline phosphatase(BAP), and wherein said marker expressed by said undifferentiatedmammalian cell contacted with said compound is upregulated by at least 3times of standard deviation from the mean of said marker expressed insaid undifferentiated mammalian cell that is not contacted with saidcompound.
 28. The method of claim 14, wherein said marker is bonealkaline phosphatase (BAP), and wherein said marker expressed by saidundifferentiated mammalian cell contacted with said compound isupregulated by at least 3 times of standard deviation from the mean ofsaid marker expressed in said undifferentiated mammalian cell that isnot contacted with said compound.
 29. The method of any one of claim 6,wherein said marker is bone alkaline phosphatase (BAP), and wherein saidmarker expressed by said undifferentiated mammalian cell contacted withsaid compound is upregulated by at least 3 times of standard deviationfrom the mean of said marker expressed in said undifferentiatedmammalian cell that is not contacted with said compound, and whereinsaid compound exhibits binding affinity of at least 10 micromolar tosaid polypeptide comprising an amino acid sequence of SEQ ID NO: 237.30. The method of any one of claim 10, wherein said marker is bonealkaline phosphatase (BAP), and wherein said marker expressed by saidundifferentiated mammalian cell contacted with said compound isupregulated by at least 3 times of standard deviation from the mean ofsaid marker expressed in said undifferentiated mammalian cell that isnot contacted with said compound, and wherein said compound exhibitsbinding affinity of at least 10 micromolar to said polypeptidecomprising an amino acid sequence of SEQ ID NO:
 237. 31. The method ofany one of claim 17, wherein said marker is bone alkaline phosphatase(BAP), and wherein said marker expressed by said undifferentiatedmammalian cell contacted with said compound is upregulated by at least 3times of standard deviation from the mean of said marker expressed insaid undifferentiated mammalian cell that is not contacted with saidcompound, and wherein said compound exhibits binding affinity of atleast 10 micromolar to said polypeptide comprising an amino acidsequence of SEQ ID NO:
 237. 32. The method of any one of claim 18,wherein said marker is bone alkaline phosphatase (BAP), and wherein saidmarker expressed by said undifferentiated mammalian cell contacted withsaid compound is upregulated by at least 3 times of standard deviationfrom the mean of said marker expressed in said undifferentiatedmammalian cell that is not contacted with said compound, and whereinsaid compound exhibits binding affinity of at least 10 micromolar tosaid polypeptide comprising an amino acid sequence of SEQ ID NO: 237.